Orientador: Ana Claudia Pavarina / Resumo: O estudo tem como objetivo avaliar a susceptibilidade ao fluconazol e a expressão de genes de virulência e envolvidos na síntese de ergosterol de cepa de Candida albicans resistente a fluconazol (ATCC 96901) presentes nas línguas de camundongos submetidos à aPDT associada a Nistatina. Para isso, colônias recuperadas de animais submetidos ao tratamento com aPDT e Nistatina (NIS) bem como a associação (NIS+aPDT e aPDT+NIS), foram armazenadas em glicerol 50% e utilizadas para o Teste de Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM). Além disso, as línguas dos animais submetidos aos tratamentos foram armazenadas em RNAlater (RNAlater Tissue Collection: RNA Stabilization Solution Protocol Ambion®) em freezer -80°C para avaliação da expressão gênica. Para realização do teste CIM, as cepas foram reativadas em placas de Ágar Sabouraud Dextrose (SDA), cultivadas em estufa 35°C por 48 h e avaliadas seguindo o método de microdiluição em caldo (CLSI) com algumas modificações. Foram realizadas diluições seriadas de fluconazol (variando de 2 a 1024 μg/ml) em meio RPMI 1640 (2 x concentrado), em suspensões (0,5x103 a 2,5x103 UFC/mL). As placas foram incubadas a 35°C e observadas quanto à presença ou ausência de crescimento após 24 h, além disso, foi realizado plaqueamento (CFM). Foram consideradas as menores concentrações de fluconazol que resultaram em inibição de pelo menos 50% do crescimento. A quantificação da expressão gênica foi realizada por meio da técni... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the susceptibility to fluconazole and the expression of virulence genes and involved in ergosterol synthesis of fluconazole resistant strain of Candida albicans (ATCC 96901) present in the mouse's thongue submitted to aPDT associated with Nystatin. For this, colonies recovered from animals submitted to treatment with aPDT and Nystatin (NIS) as well as their association (NIS + aPDT and aPDT + NIS) were stored in 50% glycerol and used for the Minimum Inhibitory Concentration Test (MIC) and Minimum Fungicidal Concentration (MFC). In addition, part of the tongue of the treated animals were stored in RNAlater (RNAlater Tissue Collection: RNA Stabilization Solution Protocol Ambion®) in -80 °C freezer for evaluation of gene expression. To accomplish the MIC test, the strains were reactivate in Sabouraud Dextrose Agar (SDA) plates, grown in a 35 °C oven for 48 h and evaluated using the broth microdilution method (CLSI) with some modifications. Serial dilutions of fluconazole (ranging from 2 to 1024 μg / ml) in RPMI 1640 medium (2x concentrate) were performed in suspensions (0.5x103 to 2.5x103 CFU / ml). The plates were incubated at 35 ° C and observed for presence or absence of growth after 24 h, plating was also performed (CFM). The lowest concentrations of fluconazole that resulted in inhibition of at least 50% growth were consider. The quantification of the gene expression was performed using quantitative Reverse Transcription-quantitative pol... (Complete abstract click electronic access below) / Mestre

Orientador: Daniela Leal Zandim-Barcelos / Resumo: A resistência bacteriana é uma ameaça real alertada pela OMS. A terapia fotodinâmica antimicrobiana (TFDA) pode ser uma das soluções para superar este desafio, uma vez que já demonstra ação antimicrobiana frente a inúmeros patógenos. A clorina-e6 (Ce6) provou ser um fotossensibilizador (FS) com potente efeito quando irradiada pela luz vermelha, contra diferentes biofilmes. No entanto, o principal pico de absorção deste FS está no espectro visível de luz azul, ainda insuficientemente investigado. Este estudo teve como objetivo avaliar o efeito da TFDA com utilização da Ce6 irradiada a 450 e 660 nm contra biofilmes relacionados à doença periodontal. Biofilmes monoespécie de Streptococcus oralis, Fusobacterium nucleatum, Porphyromonas gingivalis e Aggregatibacter actinomycetemcomitans foram desenvolvidos em condições adequadas por cinco dias. A TFDA foi realizada em diferentes concentrações de fotossensibilizador (100 e 200 µM) e comprimentos de onda de luz (450 e 660 nm), e comparada ao controle negativo (DMSO a 1%) e positivo (clorexidina a 0,2%) por análise de unidades formadoras de colônias (UFC) e microscopia confocal. O uso de luz e FS também foram testados individualmente. A maior redução bacteriana foi observada no grupo em que a TFDA foi realizada com utilização da Ce6 a 200 µM e aplicação de luz azul para todas as cepas (redução de 4,01 log10 para A. actinomycetemcomitans e redução total para P. gingivalis e S. oralis), exceto para F. nucleatum, onde o melhor resultado... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Orientador: Ana Claudia Pavarina / Resumo: O fungo Candida albicans, em situações de desequilíbrio imunológico no hospedeiro, pode se tornar um patógeno oportunista e provocar infecções. Tendo em vista a resistência que os microrganismos desenvolveram a fármacos convencionais, a Terapia Fotodinâmica Antimicrobiana (aPDT) vem apresentando resultados promissores como uma terapia alternativa. O objetivo deste estudo foi avaliar o potencial que aPDT possuiu em desenvolver resistência, tolerância ou susceptibilidade em C. albicans em associação com o Photodithazine (PDZ- 25 µg/L; 660 nm; 18 J/cm2) e Curcumina (CUR- 40 µg/L; 455 nm; 18 J/cm2) plaqueados em meio sem e com suplementação de Fluconazol (8 µg/L). C. albicans (ATCC 90028) em culturas planctônicas e biofilme foram submetidas à aplicações sucessivas de aPDT (10 aplicações), como também, a ciclos de recuperação e re-cultivo das colônias que sobreviveram ao tratamento até o momento que não houvesse mais células viáveis para a recuperação e continuação das aplicações. O plaqueamento foi realizado em placas de Ágar Sabourand Dextrose suplementadas ou não com Fluconazol, e os valores das unidades formadoras de colônia por mililitro (UFC/mL) foram determinados. O teste de produção de espécies reativas de oxigênio (ERO) foi realizado nos primeiros e últimos ciclos de aPDT. O perfil biomolecular das células sobrevivetes as aplicações 1, 4 e 7 de aPDT mediada por PDZ foi investigado através do teste de RT-qPCR. Foi observado no grupo tratamento de aPDT (P+L+) mediada por P... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The fungus Candida albicans, in situations of immune imbalance in the host, can become an opportunistic pathogen and cause infections. Because of the resistance that microorganisms have developed to conventional drugs, Antimicrobial Photodynamic Therapy (aPDT) has shown promising results as an alternative therapy. The main goal of this study was to evaluate the potential of aPDT to develop resistance in C. albicans in association with Photodithazine (PDZ-25 µg/L; 660 nm; 18 J/cm2) and Curcumin (CUR-40 µg/L; 455 nm; 18 J/cm2) grown with and without Fluconazole (8 µg/L). C. albicans (ATCC 90028), in planktonic and biofilm cultures, were submitted to successive applications of aPDT (10 applications), as well as cycles of recovery and re-cultivation of colonies that survived treatment until no longer viable cells were present for application recovery and continuation. Plating was performed on Sabourand Dextrose Agar plates supplemented or not with Fluconazole, and values of colony forming units per milliliter (CFU/mL) were determined. The reactive oxygen species (ROS) production test was performed in the first and last cycles of aPDT. The biomolecular profile of surviving cells as applications 1, 4 and 7 of PDZ-mediated aPDT was investigated using the RT-qPCR test. PDZ-mediated treatment of aPDT (P+L+) was observed to reduce approximately 6.8 log10 in planktonic cultures and approximately 6.2 log10 in C. albicans biofilm in both methodologies used. In the aPDT (C+L+) treatment gr... (Complete abstract click electronic access below) / Mestre

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Triplet-triplet annihilation upconversion (TTA-UC) is a process which converts two low energy photons into one higher-energy excited state. TTA-UC has recently received attention for its potential application to many light driven processes, such as improving efficiency in photovoltaic devices and allowing use of low-energy light sources for in vivo applications, including bioimaging, optogenetics, and photochemotherapy. Each of these applications has a different set of energetic requirements, which has created a need for a diverse library of upconverting materials. Additionally, these applications benefit from improved upconversion efficiency in solid-state, a task that has proven challenging for the traditionally solution-phase process. Macromolecular scaffolds are a promising avenue to tune the electronic communication between chromophores and control intermolecular packing in solid-state. Herein, we report the investigation of dendrimers with annihilator-functionalized termini and linear annihilator polymers as frameworks to control local annihilator concentration and communication. We find that multi-annihilator dendrimers exhibit higher upconversion yields at low concentrations compared to similar concentrations of monomer; however, higher generation dendrimers allow strong interchromophore coupling, which promotes parasitic excimer formation, decreasing relative upconversion yields. Linear annihilator copolymers with alternating anthracene and phenyl or naphthyl bridges had ground state optical properties predictive of interchromophore communication based on bridge connectivity, interchromophore length, and polymer planarity. Non-conjugated, naphthyl polymers were observed to be the most efficient at intramolecular TTA-UC in dilute solutions. In this dissertation, we will discuss current efforts in the field towards control and analysis of intramolecular TTA-UC through design of multi-annihilator macromolecules and novel annihilator scaffolds targeting underutilized regions of the electromagnetic spectrum. In Chapter 1, we list important factors to consider about improving TTA-UC and follow with discussion of reported macromolecular systems and their efforts towards intramolecular TTA-UC. Chapter 2 introduces a series of non-conjugated dendrimers functionalized with anthracene annihilators on the periphery and analyzes their upconversion capabilities as a set of macromolecules with controlled molecular structure. In Chapter 3, we investigate the effect of connectivity between annihilators in alternating co-polymer systems, discussing the impact on ground state photophysical properties and upconversion efficiency. Finally in Chapter 4, we introduce an approach for using computational analysis as a high-throughput tool for identifying potential novel annihilator molecules.

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According to the results, Pa-PDT showed inhibitory effect on MDA-MB-231 cells in vitro with an IC50 value of 0.5 muM at 24 h. Pa-PDT was demonstrated to activate intracellular mitogen activated protein kinases (MAPK) pathways via reactive oxygen species (ROS) production. Pa-PDT IS also believed to induce extracellular signal-regulated kinase (ERK)-mediated autophagy and endoplasmic reticulum stress. Pa-PDT in combination with Tamoxifen is demonstrated to exert a synergetic effect in inhibiting cancer growth. The combination treatment induces both intrinsic and extrinsic apoptosis. Regarding the direct cancer cell killing activity, two dimensional gel electrophoresis screening revealed that Pa-PDT regulates proteins which involve in human leukocyte antigen (HLA) class I-restricted antigen-processing machinery. This activation of antigen presentation was confirmed by Western blot analysis and immunostaining. Furthermore, a cross-presentation of antigen with HLA class I proteins and 70-kDa heat shock protein was found in Pa-PDT-treated cells, as shown by the fluorescent microscopic observation and immunoprecipitation assay. Moreover, the immunogenicity of breast cancer cells was increased by Pa-PDT treatment that triggered phagocytic activity by human macrophages. Our findings provide the first evidence that Pa-PDT can trigger both apoptosis and anti-tumour immunity. / Cancer is one of the most lethal diseases worldwide. Treatments of cancer comprise surgical intervention, radiotherapy or chemotherapy; however, their side effects are still need to be overcome. In order to search for anti-cancer treatments with milder side effects and higher efficiency, traditional Chinese medicine (TCM) has been investigated. Previous study in our laboratory reported that pheophorbide a (Pa), an active compound purified from Scutellaria barbata, combined with photodynamic therapy (PDT) approach produces anti-tumour effect in a wide range of human cancers. Because of the lack of protocols for curing late phase breast cancer, my project is to investigate the therapeutic potential of Pa-PDT and its action mechanism on human breast cancer. A human breast cancer cell line MDA-MB-231, which is estrogen receptor nude and resistant to a conventional breast cancer drug tamoxifen, was used as an in vitro tumour model in my study to mimic the late stage of breast cancer. / Pheophorbide a (Pa) has been proposed to be a potential photosensitizer for the photodynamic therapy of human cancer. However, the immunomodulatory effect of Pa, in the absence of irradiation, has not yet been investigated. The present study revealed that Pa possessed immunostimulating effect on a murine macrophages cell line RAW 264.7. Pa could stimulate the growth of RAW 264.7 cells with the maximal effect at 0.5 muM after 48 h of treatment, where MAPK family including c-Jun N-tenninal kinase (JNK), ERK and p38 MAPK were activated by Pa treatment in a dose-dependent manner. Moreover, the induction of interleukin-6 and tumour necrosis factor-a secretion, and the enhancement of phagocytic activity were observed in Pa-treated RAW 264.7 cells. The results were similar in Pa-treated human immune competent cells (e.g. CD4+ and CD14+ cells) at higher Pa concentrations (from 1 to 10 muM). The present work is the first report to demonstrate the potential immunomodulatory effects of Pa on immune competent cells, apart from its well-known anti-tumour activity. / Bui Xuan, Ngoc Ha. / "December 2010." / Advisers: Fung Kwok Pui; Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-144). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.