Modeling of light propagation through turbid (highly-scattering) media such as living tissue is important for a number of medical applications including diagnostics and therapeutics. This thesis studies methods of performing such simulations quickly and accurately. It begins with a formal definition of the problem, a review of solution methods, and an overview of the current state of the art in fast simulation methods encompassing both traditional software and more specialized hardware acceleration approaches (GPU, custom logic). It introduces FullMonte, the fastest mesh-based Monte Carlo software model available and highlights its novel optimiza- tions. Additionally, it demonstrates the first fully three-dimensional hardware simulator using Field-Programmable Gate Array (FPGA) custom logic, offering large (40x) power-efficiency and performance (3x) gains. Next, a plan for significant future feature enhancements and performance scale-out is sketched out. Lastly, it proposes applying the simulators developed to a number of problems relevant to current clinical and research practice.

Modeling of light propagation through turbid (highly-scattering) media such as living tissue is important for a number of medical applications including diagnostics and therapeutics. This thesis studies methods of performing such simulations quickly and accurately. It begins with a formal definition of the problem, a review of solution methods, and an overview of the current state of the art in fast simulation methods encompassing both traditional software and more specialized hardware acceleration approaches (GPU, custom logic). It introduces FullMonte, the fastest mesh-based Monte Carlo software model available and highlights its novel optimiza- tions. Additionally, it demonstrates the first fully three-dimensional hardware simulator using Field-Programmable Gate Array (FPGA) custom logic, offering large (40x) power-efficiency and performance (3x) gains. Next, a plan for significant future feature enhancements and performance scale-out is sketched out. Lastly, it proposes applying the simulators developed to a number of problems relevant to current clinical and research practice.

Antimicrobial peptides (AMPs) and photosensitizers (PS) have gained attention as potential alternatives to traditional antibiotics for the treatment of microbial infection due to the decreased likelihood for acquired resistance. However, many AMPs and PS suffer from insufficient activity, specificity, or a combination thereof. AMPs can require high concentrations for effective activity, leading to non-specific side effects and increased costs. PS, on the other hand, are quite active, but are typically hydrophobic and suffer from non-specific binding and damage to host tissues. To solve these problems, we report a novel PS-AMP construct of the soluble PS eosin Y conjugated to the selective AMP (KLAKLAK)_(2). Eosin Y has a high singlet oxygen quantum yield, which is suitable for photodynamic activity, although the solubility of eosin Y results in poor binding and activity toward membranes on its own. On the other hand, the specificity of (KLAKLAK)_(2) is high for an AMP, but could still benefit from enhanced activity at lower concentrations. The killing activity and binding specificity of eosin-(KLAKLAK)_(2) toward both bacteria and mammalian cells was assessed using microbiology, biochemistry, and fluorescence microscopy techniques. Additionally, the mechanism of eosin-(KLAKLAK)_(2) activity was investigated using liposome models to determine factors involved in binding and membrane disruption. Furthermore, novel applications of transmission electron microscopy (TEM) methods were employed to observe the photodynamic effects of eosin-(KLAKLAK)_(2) against bacteria. The PS-AMP conjugate eosin-(KLAKLAK)_(2) displays synergistic activity between PS and AMP in model liposome systems, and is capable of killing several clinically relevant bacteria, including the multi-drug resistant Acinetobacter baumannii AYE strain. Furthermore, bacterial killing is achieved in the presence of red blood cells (RBCs) and other mammalian cell lines without significant toxicity. Liposome models reveal that the lipid composition of bacteria is a potential factor responsible for the observed binding specificity and corresponding activity. Additionally, TEM methods show that eosin-(KLAKLAK)_(2) causes extensive membrane damage to both Gram positive Staph aureus and Gram negative Escherichia coli, indicating a primary cause of cell death. A model is proposed where the activities of the PS and AMP, respectively, facilitate the activity of one another, leading to enhanced membrane disruption, and effective antibacterial activity while maintaining cell selectivity.

The interaction of small molecules with DNA has been extensively studied and has produced a large catalogue of molecules that non-covalently bind to DNA though groove binding, intercalation, electrostatics, or a combination of these binding modes. Anthracene, acridine, and carbocyanine-based chromophores have been examined for their DNA binding properties and photo-reactivities. Their planar aromatic structures make them ideal chromophores that can be used to probe DNA structural interactions and binding patterns. We have studied DNA binding and photocleavgage properties of a bisacridine chromophore joined by a 2,6-bis(aminomethyl)pyridine copper-binding linker (Chapter II), a series of 9-aminomethyl anthracene chromophores (Chapters III and IV), both under conditions of high and low ionic strength, as well as a series of pentamethine linked symmetrical carbocyanine dyes (Chapter V). In Chapter II we present data showing that high ionic strength efficiently increases copper(II)-dependent photocleavage of plasmid DNA by the bisacridine based chromophore (419 nm, pH 7.0). In Chapters III and IV, using an pyridine N-substituted 9-(aminomethyl)anthracene (Chapter III), a bis-9-(aminomethyl)anthracene, and its mono 9-(aminomethyl)anthracene analogue (Chapter IV), pUC19 plasmid DNA was photo-converted to highly diffuse DNA fragments (350 nm, pH 7.0) in the presence of 150 mM NaCl and 260 mM KCl. Spectroscopic analyses suggest that the combination of salts promotes a change in DNA helical structure that initiate a switch in anthracene binding mode from intercalation to an external or groove binding interactions. The alteration in DNA structure and binding mode leads to an increase in the anthracene-sensitized production of DNA damaging reactive oxygen species. Finally, in Chapter V, pUC19 plasmid DNA is converted to its nicked circular and linear forms following irradiation of a series of pentamethine linked symmetrical carbocyanines (red light, pH 7.0). The data suggest that the relative levels of photocleavage arise from the different substituents on the nitrogen alkyl side chain and the pentamethine linker.

Ruthenium complexes show promise as light activated photodynamic therapy (PDT) prodrugs. Strained octahedral complexes were synthesized that produce a cytotoxic species upon light activation. pUC19 DNA damage in vitro experiments were carried out to determine the type of damage observed. In vivo cell experiments were carried out on the non-small lung cancer A549 cell line to determine the phototherapeutic window of the synthesized complexes. One mechanism of drug resistance via elevated levels of glutathione was addressed through in vitro binding studies carried out with UV-Vis spectroscopy and in vivo glutathione titrations in the A549 cell line. Several complexes were shown to be potential PDT agents with light-activated activities greater than cisplatin and 10-100 fold lower dark toxicities.

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%. / Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.

The purpose of this review is to compile preclinical and clinical results on phthalocyanines (Pcs) as photosensitizers (PS) for Photodynamic Therapy (PDT) and contrast agents for fluorescence imaging. Indeed, Pcs are excellent candidates in these fields due to their strong absorbance in the NIR region and high chemical and photo-stability. In particular, this is mostly relevant for their in vivo activation in deeper tissular regions. However, most Pcs present two major limitations, i.e., a strong tendency to aggregate and a low water-solubility. In order to overcome these issues, both chemical tuning and pharmaceutical formulation combined with tumor targeting strategies were applied. These aspects will be developed in this review for the most extensively studied Pcs during the last 25 years, i.e., aluminium-, zinc- and silicon-based Pcs.

As Ceratoses Actínicas (CA) são lesões hiperceratóticas, displásicas de pele. Estão comumente localizadas em áreas expostas ao sol como couro cabeludo, face e antebraços. Acredita-se que a radiação ultravioleta (RUV) cumulativa seja o maior fator etiológico, considerando-se, também, a imunossupressão e infecção pelo papilomavírus humano(HPV) fatores contribuintes importantes. Existe o potencial de uma CA se transformar em Carcinoma Espinocelular (CEC) e, dessa forma, indica-se tratamento para as referidas lesões. Terapias tradicionais como crioterapia, curetagem e eletrocoagulação, medicações tópicas como 5-fluorouracil (5-FU) são ainda habituais. Porém novas opções como terapia fotodinâmica, diclofenaco e imiquimod 5% creme apresentam boa eficácia e perfil menor de efeitos colaterais, embora possam representar custo maior. A Terapia Fotodinâmica (TFD) envolve o uso de um agente fotossensibilizante, oxigênio e luz de comprimento de onda específico para causar morte celular. O fotossensibilizante geralmente utilizado é o ALA (ácido aminodeltalevulínico) ou seu éster metilaminolevulinato (MAL). No tecido lesionado, esses são convertidos em porfirinas fotoativas (PFAs) por enzimas da via biossintética do heme. A ativação é realizada por meio de luzes de comprimentos de onda que variam de 405 nm a 635 nm. Células displásicas ou neoplásicas produzem maior quantidade de porfirinas que os queratinócitos normais, sendo destruídas durante a aplicação da luz. O imiquimod 5% creme é um imunomodulador que estimula a resposta imune inata através da indução, síntese e liberação de citocinas. Isso resulta em efeitos antitumorais e antivirais indiretos. Seu uso tópico é eficaz e liberado para o tratamento de CA, Carcinoma Basocelular (CBC) superficial, Doença de Bowen (DB) e verrugas vulgares. Este trabalho tem como objetivo comparar duas recentes opções de tratamento para CA, a Terapia Fotodinâmica com metilaminolevulinato e o imiquimod 5% creme, por não existirem, na literatura atual, estudos comparativos dessas duas opções de tratamento. Foram selecionadas 12 pacientes com CAs que foram submetidas primeiramente à 1 sessão de TFD com MAL num lado da face e, 1 mês após, iniciaram o tratamento com imiquimod 5% creme aplicado no lado contralateral, duas vezes na semana, durante 16 semanas. A randomização foi realizada para determinar a hemiface para cada tratamento. Na primeira semana após a realização da TFD e mensalmente durante o tratamento com imiquimod, as pacientes foram avaliadas em relação aos efeitos colaterais dos tratamentos. Seis meses após entrarem no estudo, ambos os tratamentos foram analisados por um investigador cego para sua eficácia, tolerabilidade e seu resultado cosmético. Previamente ao tratamento as pacientes apresentaram um total de 245 lesões de CAs, sendo 120 lesões no lado submetido à TFD e 125 no lado tratado com imiquimod. Após o tratamento o número total de lesões diminuiu para 34 no lado tratado com TFD e para 30 no lado tratado com imiquimod, respectivamente. Não foram observadas diferenças estatisticamente significativas na eficácia de ambos os tratamentos e na frequência de efeitos colaterais. Entretanto, os pacientes, significativamente preferiram o tratamento com a terapia fotodinâmica, talvez pela rapidez do método, comparado com a aplicação tópica do creme de imiquimod a 5%. / Background: Actinic keratosis (AK) represents an initial process that may lead to in situ or invasive squamous cell carcinoma. The importance of its early diagnosis and treatment is well-established. There are several effective options available for the treatment of actinic keratosis, including topical imiquimod 5% cream and photodynamic therapy (PDT). Objetive: To compare the efficacy and patient preference between topical MAL-PDT and imiquimod 5% cream for the treatment of AK. Methods: Twelve patients, with a total of 245 lesions, underwent treatment with MAL-PDT and imiquimod 5% cream. Randomization was performed to determine the hemiface (right or left) for each therapy. First, patients were submitted to MAL-PDT. After one month, they started to use imiquimod on the opposite side of the face, twice a week, for 16 weeks. Six months after entering the study, both treatments were analyzed by a blinded investigator for their effectiveness, tolerability and cosmetic result. Results: Both treatments showed a good therapeutic response. 72% of the lesions treated with MAL-PDT were completely cleared, and 76% of those treated with imiquimod. The mean size of the residual lesions after the treatments was similar. Ten patients (83%) preferred MAL-PDT rather than imiquimod. (p: 0.03). Conclusions: Both MAL-PDT and imiquimod are effective in clearing AKs. Our results showed similar efficacy and good cosmetic outcomes with both treatments. However, a significant percentage of the subjects preferred MAL – PDT.

A Terapia Fotodinâmica (TFP) é uma técnica para tratamento de câncer que usa um fotossensibilizador (FS) na presença de luz e oxigênio gerando espécies altamente reativas de oxigênio que levam as células tumorais à morte. <br />Neste trabalho foi realizado um estudo comparativo com três FSs: Photogem&reg; (PG), um derivado de hematoporfirina que está sendo usado em TFD no Brasil; Photodithazine&reg; (PZ), um derivado hidrossolúvel de mono-L-aspartil clorina, que está na fase clínica para aprovação e Hipericina (HY), um pigmento fotoativo encontrado na planta Hypericum perforatum e usado na medicina popular que está sendo considerado como um promissor agente fotodinâmico para o tratamento de tumores. Este estudo utilizou uma Hipericina sintetizada no Brasil e diversos parâmetros para comparar os três FSs: a concentração inibitória média (IC50) em linhagens celulares; a constante de velocidade de fotoxidação da albumina de soro bovino na presença dos FSs e luz determinada pelo decréscimo na fluorescência da BSA em 340 nm; a fotoxidação do ácido úrico acompanhada pelo decréscimo da banda característica do ácido úrico em 290 nm após irradiação na presença dos FSs como uma estimativa indireta do rendimento quântico de formação de oxigênio singlete (&Delta;&Phi;); o rendimento quântico de fluorescência utilizando rodamina B como padrão; a acumulação dos FSs em células em função do tempo de incubação e a estimativa da quantidade de radicais livres formados após irradiação através da técnica de captura de spins. Todos os resultados obtidos evidenciam uma maior eficiência fotodinâmica da HY seguida pelo PZ e depois por Photogem e, portanto sugerem a Hipericina como o FS de maior potencial para utilização em Terapia Fotodinâmica. / Photodynamic Therapy (PDT) is a technique for the cancer treatment that uses a photosensitizer (FS) in the presence of light and oxygen which combined are able to generate highly reactive oxygen species that lead to tumor cells death. <br />In this investigation, a comparative study with three FSs: Photogem &reg; (PG), a hematoporphyrin derivative being used in PDT in Brazil; Photodithazine &reg; (PZ), a soluble derivative of mono-L-aspartyl chlorin, which is in clinical phase for approval and Hypericin (HY), a photoactive pigment found in the plant Hypericum perforatum and used in popular medicine that is being considered as a promising agent for photodynamic treatment of tumors. The present study used a Hypericin synthesized in Brazil and several parameters to compare these three FSs: the mean inhibitory concentration (IC50) in cell lines; the rate constant for the photooxidation of bovine serum albumin in the presence of light and the FSs determined by the decrease in the fluorescence of BSA at 340 nm; the photooxidation of uric acid assessed by the decrease of the characteristic band of uric acid at 290 nm after irradiation in the presence of the FSs as an indirect estimate of the quantum yield of formation of singlet oxygen (&Delta;&Phi;); the quantum yield of fluorescence using rhodamine B as a standard; the accumulation of FSs in cells as a function of the incubation time, and the estimative of the produced free radicals after irradiation by the technique of spin trapping. All the results show a higher photodynamic efficiency of HY followed by PZ and then by Photogem suggesting Hypericin as the FS with the greatest potential for use in Photodynamic Therapy.

Made available in DSpace on 2014-06-11T19:27:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-06-09Bitstream added on 2014-06-13T18:31:37Z : No. of bitstreams: 1 martins_js_me_sjc.pdf: 608313 bytes, checksum: e32f819f13bdf398082ecb9e208a1b5a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O objetivo deste estudo foi avaliar os efeitos da terapia fotodinâmica sobre a patogenicidade de C. albicans e C. dubliniensis na candidose bucal em ratos. Foram utilizados 96 ratos (Rattus norvegicus, Albinus, Wistar), sendo que 80 animais foram submetidos à indução de candidose experimental no dorso da língua por C. albicans ou C. dubliniensis e 16 ratos não foram infectados por Candida. Após 5 dias da indução da candidose, foram realizados diferentes tratamentos: laser e azul de metileno como fotossensibilizador (Grupo L+F+), apenas laser (Grupo L+F-), apenas fotossensibilizador (Grupo L-F+) e grupo sem laser e fotossensibilizador (Grupo L-F-). Após 1 dia, amostras do dorso da língua foram coletadas e semeadas em ágar Sabouraud para contagem de UFC/mL recuperadas da cavidade bucal. Os animais foram submetidos à eutanásia para análise microscópica do dorso da língua. Foi avaliada também a capacidade de produção de fosfolipase e proteinase por C. albicans e C. dubliniensis. Os resultados demonstraram que a quantidade de C. albicans recuperadas da cavidade bucal dos ratos foram semelhantes entre os quatro grupos estudados. C. dubliniensis não foi recuperarada da cavidade bucal dos ratos estudados. Na análise microscópica do dorso da língua, observou-se a presença de várias lesões teciduais induzidas por C. albicans, sendo que no grupo L+F+ essas lesões foram menores em relação aos demais grupos estudados. C. dubliniensis exibiu lesões teciduais discretas sem diferença estatística entre os grupos. Em relação aos fatores de patogenicidade de C. albicans, o grupo L+F+ produziu menor quantidade de fosfolipase e proteinase em relação aos demais grupos. C. dubliniensis produziu menor quantidade de fosfolipase em relação à C. albicans, sem diferença entre os 4 grupos estudados. Entretanto, C. dubliniensis não foi capaz de produzir proteinase... / The objective was to evaluate the effects of photodynamic therapy on the pathogenicity of C. albicans and C. dubliniensis in oral rats candidosis. Were evaluated 96 rats, while 80 were subjected to candidosis experimental induction on the tongue dorsum by C. albicans or C. dubliniensis and 16 rats were not infected. After 5 days of induction candidosis, different treatments were performed: laser and methylene blue as photosensitizers (Group L+F+), laser only (Group L+F-), only photosensitizers (Group L-F+) and without laser neither photosensitizers group (Group L-F). After 1 day, samples of the tongue dorsum were collected and sown on Sabouraud agar for counts of UFC/mL recovered from the oral cavity. The animals were submitted to euthanasia for microscopic analysis. It also evaluated the production capacity of a proteinase and phospholipase C. albicans and C. dubliniensis. The results showed that the amount of C. albicans recovered from rats oral cavity were similar among the four groups. To recover C. dubliniensis of the oral cavity from studied rats was not possible. In microscopic examination of tongue dorsum, was seen the presence of multiple tissue lesions induced by C. albicans, whereas in group L+ F+ these lesions were smaller than in other groups. C. dubliniensis showed slight tissue lesions with no statistic difference between groups. Regarding the factors of pathogenicity of C. albicans, the group L+F+ produced smaller amounts of phospholipase and proteinase compared to other groups. C. dubliniensis produced fewer quantitiy of phospholipase related to C. albicans, without difference between the 4 (four) groups studied. However, C. dubliniensis was not able to produce proteinase. It was concluded that the development of experimental candidosis by C. dubliniensis was less than that induced by C. albicans. Photodynamic therapy was effective on treatment... (Complete abstract click electronic access below)