Hoi, Wan Heng. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 212-245). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Cheung, Ka Yan. / "Aug 2010." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 157-167). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgments --- p.v / Table of Contents --- p.vi / List of Figures --- p.x / List of Tables --- p.xi / Abbreviations --- p.xii / Chapter Chapter1 --- General Introduction --- p.1 / Chapter 1.1 --- Cancer epidemiology and managements --- p.2 / Chapter 1.2 --- Photodynamic therapy (PDT) as cancer treatment --- p.7 / Chapter 1.3 --- Pheophorbide a (Pa) as a photosensitizer for PDT --- p.13 / Chapter 1.4 --- Aim of study --- p.15 / Chapter Chapter2 --- The anti-proliferative effect of pheophorbide a- mediated photodynamic therapy on human breast adenocarcinoma cell line MCF-7 --- p.17 / Chapter 2.1 --- Introduction / Chapter 2.1.1 --- Cell cycle regulation --- p.18 / Chapter 2.1.2 --- Growth arrest and DNA damage inducible (GADD) genes as cell cycle regulators --- p.22 / Chapter 2.2 --- Materials and Methods / Chapter 2.2.1 --- Materials / Chapter 2.2.1.1 --- Cell line --- p.29 / Chapter 2.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.29 / Chapter 2.2.1.3 --- Gene expression assay reagents --- p.30 / Chapter 2.2.1.4 --- Reagents and buffers for Western blotting --- p.32 / Chapter 2.2.1.5 --- Cell cycle analysis reagents --- p.35 / Chapter 2.2.2 --- Methods / Chapter 2.2.2.1 --- Cell line propagation and subculture --- p.36 / Chapter 2.2.2.2 --- Whole-transcript expression micro array analysis --- p.37 / Chapter 2.2.2.3 --- GADD genes expression assay- RT-PCR --- p.37 / Chapter 2.2.2.4 --- Cell cycle analysis --- p.40 / Chapter 2.2.2.5 --- Western Blotting --- p.41 / Chapter 2.2.2.6 --- Statistical analysis --- p.43 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Effect of Pa-PDT on GADD genes expression by whole-transcript expression microarray analysis --- p.44 / Chapter 2.3.2 --- Effect of Pa-PDT on GADD genes expression by RT-PCR --- p.46 / Chapter 2.3.3 --- Temporal change in the cell cycle profile after Pa-PDT --- p.48 / Chapter 2.3.4 --- Effect of Pa-PDT on cell cycle associated proteins --- p.65 / Chapter 2.4 --- Discussion --- p.67 / Chapter Chapter3 --- Development of drug resistance in human breast adenocarcinoma cell line MDA and the circumvention by pheophorbide a-mediated photodynamic therapy --- p.77 / Chapter 3.1 --- Introduction / Chapter 3.1.1 --- Clinical Importance of multidrug resistance (MDR) --- p.78 / Chapter 3.1.2 --- Mechanisms of MDR --- p.78 / Chapter 3.1.3 --- Development of MDR cell lines --- p.82 / Chapter 3.1.4 --- Reversal of MDR by P-glycoprotein modulators --- p.83 / Chapter 3.1.5 --- Therapeutic potential of Pa-PDT in treating MDR cancers --- p.83 / Chapter 3.2 --- Materials and Methods / Chapter 3.2.1 --- Materials / Chapter 3.2.1.1 --- Cell line --- p.85 / Chapter 3.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.85 / Chapter 3.2.1.3 --- Cell viability assay reagents --- p.85 / Chapter 3.2.1.4 --- Gene expression assay reagents --- p.86 / Chapter 3.2.2 --- Methods / Chapter 3.2.2.1 --- Cell line propagation and subculture --- p.87 / Chapter 3.2.2.2 --- Drug-resistance development --- p.88 / Chapter 3.2.2.3 --- Measurement of cell viability - MTT reduction assay --- p.88 / Chapter 3.2.2.4 --- ABCB1 expression assay- RT-PCR --- p.89 / Chapter 3.2.2.5 --- Doxorubicin uptake assay --- p.91 / Chapter 3.2.2.6 --- Pheophorbide a uptake assay --- p.91 / Chapter 3.2.2.7 --- Statistical analysis --- p.92 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Cytotoxicity of doxorubicin on MDA and MDA-R cells --- p.93 / Chapter 3.3.2 --- mRNA expression of ABCB1 (P-glycoprotein) in MDA and MDA-R cells --- p.96 / Chapter 3.3.3 --- Doxorubicin uptake by MDA and MDA-R cells --- p.98 / Chapter 3.3.4 --- Circumvention of drug resistance in MDA-R cells by Pa-PDT --- p.102 / Chapter 3.3.5 --- Pheophorbide a uptake by MDA and MDA-R cells --- p.104 / Chapter 3.4 --- Discussion --- p.106 / Chapter Chapter4 --- Synergistic anti-proliferation of pheophorbide a-mediated photodynamic therapy and doxorubicin on multidrug resistant uterine sarcoma cell line Dx5 --- p.113 / Chapter 4.1 --- Introduction / Chapter 4.1.1 --- Clinical limitations of doxorubicin as chemotherapeutic drug --- p.114 / Chapter 4.1.2 --- Clinical limitations of photodynamic therapy --- p.115 / Chapter 4.1.3 --- Combination therapy with Dox and Pa-PDT --- p.117 / Chapter 4.1.4 --- Uterine sarcoma cell line Dx5 as in vitro model for combination therapy --- p.118 / Chapter 4.2 --- Materials and Methods / Chapter 4.2.1 --- Materials / Chapter 4.2.1.1 --- Cell line --- p.120 / Chapter 4.2.1.2 --- "Cell culture medium, supplements and other reagents" --- p.120 / Chapter 4.2.1.3 --- Anti-cancer drugs --- p.121 / Chapter 4.2.1.4 --- "ROS inhibitor, α-tocopherol" --- p.121 / Chapter 4.2.1.5 --- Cell viability assay reagents --- p.122 / Chapter 4.2.1.6 --- P-glycoprotein activity assay reagents --- p.122 / Chapter 4.2.2 --- Methods - / Chapter 4.2.2.1 --- Cell line propagation and subculture --- p.123 / Chapter 4.2.2.2 --- Cell viability assay --- p.123 / Chapter 4.2.2.3 --- P-glycoprotein activity assay --- p.124 / Chapter 4.2.2.4 --- Statistical analysis --- p.125 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Combination therapy of Pa-PDT and doxorubicin in Dx5 cells --- p.126 / Chapter 4.3.2 --- Effect of α-tocopherol on the synergism between Pa-PDT and doxorubicin in Dx5 cells --- p.129 / Chapter 4.3.3 --- Effect of Pa-PDT on P-glycoprotein activity in Dx5 cells --- p.132 / Chapter 4.3.4 --- Combination therapy of Pa-PDT and doxorubicin in SA cells --- p.138 / Chapter 4.4 --- Discussion --- p.141 / Chapter Chapter5 --- General Discussion --- p.148 / Chapter 5.1 --- Pa-PDT induced growth arrest and DNA fragmentation in breast cancer MCF-7 cells --- p.149 / Chapter 5.2 --- Circumvention of doxorubicin resistance by Pa-PDT in breast cancer MDA cells --- p.151 / Chapter 5.3 --- Synergistic anti-proliferation of Pa-PDT and doxorubicin on uterine sarcoma cell line Dx5 --- p.151 / Chapter 5.4 --- Clinical implication --- p.153 / Chapter 5.5 --- Conclusions and future perspectives --- p.153 / References --- p.157

Photodynamic therapy (PDT) is a multimodality cancer treatment available for the palliation or eradication of systemic and cutaneous malignancies. In this thesis, the application of PDT is for the treatment of non-melanoma skin cancer (NMSC). While PDT has a well-documented track record, there are, at this time no significant indicators to suggest the superiority of one treatment regime over the next. The motivation for this work is to provide additional evidence pertaining to PDT treatment variables, and to assist in optimising PDT treatment regimes. One such variable is the treatment light dose. Determining the light dose more accurately would assist in optimising treatment schedules. Furthermore, choice of photosensitiser pro-drug type and application times still lack an evidence base. To address issues concerning treatment parameters, fluorescence spectroscopy – a valuable optical diagnostic technique – was used. Monitoring the in vivo PpIX fluorescence and photobleaching during PDT was employed to provide information pertaining to the progression of treatment. This was demonstrated by performing a clinical study at the Photobiology Unit, Ninewells Hospital and Medical School, Dundee. Two different photosensitiser pro-drugs – either 5-aminolaevulinic acid (ALA) or its methyl ester (MAL) – were investigated and based on the fluorescence and pain data recorded both may be equally suitable for topical PDT. During PDT, surface fluorescence is observed to diminish with time – due to photobleaching – although cancerous cells may continue to be destroyed deep down in the tissue. Therefore, it is difficult to ascertain what is happening at depth in the tumour. This raised the questions; How long after surface PpIX fluorescence has diminished is the PDT treatment still effective and to what depths below the surface is effective treatment provided? In order to address these important questions, a three-dimensional (3D) Monte Carlo radiation transfer (MCRT) model was used to compute the light dose and the ¹O₂ production within a tumour, and the PpIX fluorescence emission from the tumour. An implicit dosimetry approach based on a single parameter – fluorescence photobleaching – was used in order to determine the ¹O₂ generation, which is assumed to be related to tissue damage. Findings from our model recommended administering a larger treatment light dose, advocating an increase in the treatment time after surface PpIX fluorescence has diminished. This increase may ultimately assist in optimising PDT treatment regimes, particularly at depth within tumours.

Chan, Man Hung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 111-126). / Abstract also in Chinese. / Examination Committee List --- p.ii / Declaration --- p.iii / Acknowledgements --- p.iv / 摘要（Abstract in Chinese) --- p.vi / Abstract --- p.viii / List of Abbreviations --- p.x / List of Figures and Tables --- p.xii / Table of Content --- p.xiv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Background of photodynamic therapy (PDT) --- p.2 / Chapter 1.1.1 --- History of PDT --- p.2 / Chapter 1.1.2 --- Photochemistry --- p.3 / Chapter 1.1.3 --- Principal stages of PDT --- p.5 / Chapter 1.1.4 --- Light sources of PDT --- p.6 / Chapter 1.2 --- Anti-tumor effect of PDT --- p.8 / Chapter 1.2.1 --- Mode of cell death --- p.8 / Chapter 1.2.2 --- PDT-induced anti-tumor immunity --- p.9 / Chapter 1.3 --- Clinical applications of PDT --- p.11 / Chapter 1.3.1 --- Photofrin® --- p.11 / Chapter 1.3.2 --- Clinical applications of PDT --- p.13 / Chapter 1.3.3 --- Challenges of PDT for clinical applications --- p.15 / Chapter 1.4 --- The development of new photosensitizers --- p.16 / Chapter 1.4.1 --- Targeted PDT --- p.16 / Chapter 1.4.2 --- Phthalocyanine --- p.18 / Chapter 1.5 --- Objective of my study --- p.21 / Chapter Chapter 2 --- Materials and Methods --- p.23 / Chapter 2.1 --- Synthesis of glucosylated silicon(IV) phthalocyanine (SiPcGlu) --- p.24 / Chapter 2.2 --- In vitro studies --- p.24 / Chapter 2.2.1 --- Cell line and culture conditions --- p.24 / Chapter 2.2.2 --- Photodynamic treatment --- p.25 / Chapter 2.2.3 --- Cell viability assay --- p.27 / Chapter 2.2.4 --- Light dose effect on the photocytotoxicity of SiPcGlu-PDT --- p.27 / Chapter 2.2.5 --- Determination of reactive oxygen species (ROS) production by SiPcGlu-PDT --- p.29 / Chapter 2.2.6 --- Effect of antioxidants on the photocytotoxicity of SiPcGlu-PDT --- p.29 / Chapter 2.2.7 --- Determination of ROS production after SiPcGlu-PDT --- p.30 / Chapter 2.2.8 --- Glucose competitive assay --- p.30 / Chapter 2.2.9 --- Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay --- p.30 / Chapter 2.2.10 --- DNA fragmentation analysis by gel electrophoresis --- p.31 / Chapter 2.2.11 --- Annexin-V & propidium iodide staining assay --- p.32 / Chapter 2.2.12 --- Subcellular localization studies --- p.33 / Chapter 2.2.13 --- Detection of mitochondrial superoxide production --- p.34 / Chapter 2.2.14 --- Assessment of mitochondrial membrane potential --- p.34 / Chapter 2.2.15 --- Caspase-3 activity assay --- p.35 / Chapter 2.2.16 --- "Western blot analyses for cytochrome c, caspase-3, PARP and glucose-regulated protein 78 (GRP78)" --- p.36 / Chapter 2.2.17 --- Ca2+ release from endoplasmic reticulum (ER) --- p.37 / Chapter 2.3 --- In vivo studies --- p.37 / Chapter 2.3.1 --- HT29 tumor-bearing nude mice model --- p.37 / Chapter 2.3.2 --- In vivo photodynamic treatment --- p.39 / Chapter 2.3.3 --- Biodistribution of SiPcGlu --- p.39 / Chapter 2.3.4 --- Assay for plasma enzyme activities --- p.40 / Chapter 2.4 --- Statistical analysis --- p.41 / Chapter Chapter 3 --- Results --- p.42 / Chapter 3.1 --- In vitro studies --- p.43 / Chapter 3.1.1 --- SiPcGlu-PDT induced cytotoxicity on HT29 cells --- p.43 / Chapter 3.1.2 --- Light dose effect on cytotoxicity by SiPcGlu-PDT --- p.46 / Chapter 3.1.3 --- SiPcGlu-PDT induced ROS production --- p.48 / Chapter 3.1.4 --- SiPcGlu-PDT induced cell death through Type I and II photoreactions --- p.48 / Chapter 3.1.5 --- ROS production after SiPcGlu-PDT --- p.51 / Chapter 3.1.6 --- Glucose competitive Assay --- p.55 / Chapter 3.1.7 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.57 / Chapter 3.1.8 --- Subcellular localization of SiPcGlu --- p.61 / Chapter 3.1.9 --- SiPcGlu-PDT induced mitochondrial changes --- p.66 / Chapter 3.1.10 --- SiPcGlu-PDT induced caspase activation --- p.68 / Chapter 3.1.11 --- SiPcGlu-PDT increased expression of ER chaperone GRP78 --- p.72 / Chapter 3.1.12 --- SiPcGlu-PDT induced release of Ca2+ from ER --- p.72 / Chapter 3.2 --- In vivo studies --- p.75 / Chapter 3.2.1 --- In vivo photodynamic activities --- p.75 / Chapter 3.2.2 --- Tissue distribution of SiPcGlu --- p.77 / Chapter 3.2.3 --- Analysis of intrinsic toxicity --- p.77 / Chapter Chapter 4 --- Discussion --- p.80 / Chapter 4.1 --- Physical Properties of SiPcGlu --- p.81 / Chapter 4.2 --- In vitro studies --- p.82 / Chapter 4.2.1 --- SiPcGlu-PDT exhibits a high potency in killing HT29 cells --- p.82 / Chapter 4.2.2 --- ROS production is responsible for the cytotoxic effect of SiPcGlu-PDT --- p.83 / Chapter 4.2.3 --- SiPcGlu-PDT induced apoptosis in HT29 cells --- p.85 / Chapter 4.2.4 --- SiPcGlu is localized in various membranous organelles --- p.87 / Chapter 4.2.5 --- SiPcGlu-PDT induced mitochondria-mediated apoptosis --- p.89 / Chapter 4.2.6 --- SiPcGlu-PDT induced ER stress --- p.93 / Chapter 4.3 --- In vivo studies --- p.96 / Chapter 4.3.1 --- SiPcGlu failed to target to tumor tissues --- p.96 / Chapter 4.3.2 --- SiPcGlu-PDT induced retardation in tumor growth --- p.99 / Chapter 4.3.3 --- SiPcGlu is a safe photosensitizer for PDT --- p.101 / Chapter Chapter 5 --- Conclusion and Future Perspectives --- p.103 / Chapter 5.1 --- Conclusion --- p.104 / Chapter 5.2 --- Future Perspectives --- p.106 / Chapter 5.2.1 --- In vitro studies --- p.106 / Chapter 5.2.1.1 --- Lysosomal pathway to cell death --- p.106 / Chapter 5.2.2 --- In vivo studies --- p.107 / Chapter 5.2.2.1 --- Pharmacokinetic studies --- p.107 / Chapter 5.2.2.2 --- Eradication of HT29 tumor by repeated dose of SiPcGlu --- p.108 / Chapter 5.2.2.3 --- SiPcGlu-PDT-induced anti-tumor immunity --- p.108 / Chapter 5.2.2.4 --- Enhancement of tumor selectivity by conjugating with biomolecules --- p.109 / References --- p.110

Submitted in fulfillment of the requirements for the degree of Doctor of Philosophy: Biotechnology, Durban University of Technology, Durban, South Africa, 2015. / Photodynamic therapy (PDT) is emerging as a viable alternative to invasive anti-cancer treatment regimens such as surgery, chemotherapy or radiotherapy. A series of metal – based phthalocyanine complexes have been discovered that may be used as a drug or photosensitizer in photodynamic therapy for the treatment of cancers. During photodynamic therapy the photosensitizer is administrated intravenously or topically to the patient before laser treatment at an appropriate wavelength is delivered to the cancerous site to activate the photosensitizer. The activated photosensitizer will react with oxygen typically present in the cancerous tissue to produce reactive oxygen species for the eradication of the cancerous tissue. This is the first study where gallium (GaPcCl), indium (InPcCl) and iron (FePcCl) Pc chloride complexes were used for photodynamic research. These metal – based phthalocyanine complexes were investigated using different cancer cell lines (Caco-2, MCF-7, melanoma and A549). Also, the baseline cellular uptake and photodynamic effect of these complexes were established on healthy normal cells (human fibroblast cells). Fluorescent spectrophotometry showed that all three photosensitizers accumulated in a time-dependent manner in Caco-2, MCF-7, melanoma and A549 cancer cells, as well as in healthy normal fibroblast cell in amounts which increased over a period of 24 hours, with emission peaking at 24 hours for all cell lines. Dark toxicity effects and photodynamic therapy efficacy were established with a MTT assay. High concentrations of inactive GaPcCl, InPcCl and FePcCl was toxic to Caco-2, melanoma, A549 and fibroblast cells. However, all three photosensitizers were in its inactive state at low and high photosensitizing concentrations were highly toxic to MCF-7 cancer cells. On the other hand, in vitro photodynamic therapy treatment with both low and high concentrations of GaPcCl, InPcCl and FePcCl were observed to be potently cytotoxic towards all four cancer cell lines upon exposure to laser light for 22 seconds (2.5 J/cm2), 39 seconds (4.5 J/cm2) and 74 seconds (8.5 J/cm2). These results revealed that all three photosensitizers reacts to photodynamic therapy in a concentration-dependent (photosensitizer) and dose-dependent (light dose/time) manner. At 24 hours after photodynamic therapy, the most effective treatment parameters were laser treatment for 74 seconds with FePcCl concentrations from 60 µg/ml - 100 µg/ml which resulted in 0% cell survival of Caco-2 cancer cells. A short laser treatment time of 74 seconds for activation of FePcCl (20 µg/ml) resulted in 0% cell survival of MCF-7 cancer cells. Similarly, FePcCl (40 µg/ml - 100 µg/ml) activated for 22 seconds, 39 seconds and 74 seconds resulted in 100% cell death of A549 cancer cells. Photodynamic therapy treatment with GaPcCl and InPcCl were very effective in reducing the cell viability of melanoma cancer cells. Healthy normal fibroblast cells survived in vitro photodynamic therapy treatment with all three photosensitizers much better than the cancer (Caco-2, MCF-7, melanoma and A549) cells. This confirms the previously reported results that photosensitizers such as phthalocyanines and its metal-based complexes preferentially accumulate in cancer cells than normal healthy cells. All three photosensitizers localized in mitochondria and lysosomes of the Caco-2, MCF-7 and A549 cancer cells. In melanoma cancer cells InPcCl also localized in the mitochondria and lysosome, but GaPcCl and FePcCl localized in mitochondria only. Apoptosis was identified via microscopical and flow cytometric investigations, as the dominant mode of cell death induced by GaPcCl, InPcCl and FePcCl mediated photodynamic therapy in cancer cell lines tested. Therefore, this study concludes that GaPcCl, InPcCl and FePcCl are effective photosensitizers for the in vitro PDT treatment of cancer cells. The effective in vitro PDT treatment for each cell line was dependent on the photosensitizer concentration and illumination period for each of the different photosensitizers. / D

Orientador: Alessandra Nara de Souza Rastelli / Resumo: A inativação fotodinâmica é descrita como terapêutica promissora na inativação de micro-organismos bucais. A curcumina é um fotossensibilizador com característica hidrofóbica, e que pode comprometer a sua eficiência. Dessa forma, torna-se relevante o desenvolvimento de estudos que verifiquem o seu efeito quando conjugada a micelas poliméricas. O objetivo deste estudo foi avaliar a efetividade da inativação fotodinâmica em biofilme de Streptococcus mutans utilizando-se curcumina, conjugada ou não, a micelas como fotossensibilizador, irradiado por luz LED. Realizou-se a caracterização das micelas poliméricas nas concentrações de 0.1 e 0.5% de curcumina, por meio do Potencial Zeta, determinação do espalhamento de luz dinâmico, microscopia eletrônica de transmissão e a investigação dos mecanismos envolvidos na reação fotodinâmica. Após, foi selecionada a melhor concentração a ser utilizada na inativação fotodinâmica sobre biofilme de Streptococcus mutans. As concentrações inibitória mínima (CIM) e bactericida mínima (CBM) foram determinadas. Foram induzidos em placa de 96 poços biofilmes single espécie. Os biofilmes foram irradiados uniformemente com sistema de iluminação a LED (Biotable, MMO) em comprimento de onda de 460 nm com dose/densidade de energia de 15 J/cm2 . Unidades formadoras de colônia (UFC/mL), a partir da CIM e CBM foram obtidas. Por meio de microscopia confocal utilizando-se corante BacLight® LIVE/DEAD foi avaliado a viabilidade celular nos biofilmes. Diferente... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Photodynamic inactivation is described as promising therapy in the inactivation of oral microorganisms. Curcumin is a hydrophobic photosensitizer, which can compromise its efficiency. Thus, the development of studies that verify its effect when conjugated to polymeric micelles becomes relevant. The objective of this study was to evaluate the effectiveness of photodynamic inactivation in Streptococcus mutans biofilm using curcumin, conjugated or not, to micelles as photosensitizer, irradiated by LED light. The characterization of the polymeric micelles in the 0.1% and 0.5% concentrations of curcumin was carried out by Zeta Potential, determination of dynamic light scattering, transmission electron microscopy and investigation of the mechanisms involved in the photodynamic reaction. After that, the best concentration to be used in photodynamic inactivation on Streptococcus mutans biofilm was selected. Minimum inhibitory concentrations (MIC), minimal bactericidal (MBC) were determined. Single-species biofilms were induced in 96-well plate. The biofilms were uniformly irradiated with a LED illumination system (Biotable, MMO) at wavelength of 460 nm with dose or energy density of 15 J/cm2. Colony forming units (CFU / mL) from CIM and CBM were obtained. By means of confocal microscopy using BacLight® LIVE/DEAD dye, cell viability in biofilms was evaluated. Different groups were analyzed: FSM+L+ (Photosensitizer conjugate micelles Pluronic + Light), FSD+L+ (Photosensitizer in 10% DM... (Complete abstract click electronic access below) / Mestre

O objetivo do estudo foi avaliar o efeito de repetidas aplicações da Terapia Fotodinâmica antimicrobiana (TFDa) adjuvante ao tratamento periodontal não cirúrgico em pacientes fumantes. Foram selecionados 20 indivíduos fumantes com diagnóstico clínico de periodontite crônica. O estudo foi do tipo boca dividida, sendo um lado utilizado a TFDa associada à raspagem e alisamento radicular (RAR) e no outro apenas RAR. Foi utilizado laser com 660nm de comprimento de onda associada ao fotosensibilizador fenotiazida e as aplicações foram realizadas em 4 episódios (dias 0, 2, 7 e 14). Todos os pacientes foram acompanhados por 90 dias. As avaliações clínicas de índice de placa (IP), profundidade de sondagem (PS), nível clínico de inserção (NCI), sangramento à sondagem (SS) e sangramento marginal (SM) foram realizadas no baseline, 0, 30 e 90 dias após a execução do tratamento. Foi realizada análise microbiológica para contagem de 40 espécies bacterianas da amostra subgengival da placa bacteriana (Checkerboard DNA-DNA hybridization). Níveis das citocinas do fluido crevicular gengival foram avaliados (Interleucina-1β, Interleucina-10 e Fator de Necrose Tumoral Alpha). Os dados obtidos foram analisados estatisticamente. Em geral não foram observadas diferenças estatisticamente significantes entre os grupos (p &lt0,05) nos parâmetros clínicos, microbiológicos e imunológicos 90 dias após o tratamento. O tratamento periodontal com RAR + TFDa em múltiplos episódios não promoveu benefícios adicionais à RAR no tratamento não cirúrgico da periodontite crônica em fumantes sem o uso de antibióticos sistêmicos / The aim of this study was to investigate the additional influence of multiple applications of antimicrobial Photodynamic Therapy (aPDT) in smokers without use of systemic antibiotics. Twenty smokers with chronic periodontitis were treated in a split-mouth design study with aPDT adjunct to Scaling and Root Planing (SRP) or SRP only. aPDT was performed by using a laser light source with 660 nm wavelength associated with a photosensitizer. The applications were performed in four episodes (at days 0, 2, 7 and 14). All patients were monitored for 90 days. Plaque index, probing depth, clinical attachment level, bleeding on probing and marginal bleeding were performed at baseline, 0, 30 and 90 days after the SRP. Counts of 40 subgingival species in plaque samples were monitored (Checkerboard DNA-DNA hybridization). Gingival crevicular fluid and subgingival plaque samples were collected (Interleukin 1β, Interleukin 10 e Tumor necrosis factor. Data obtained were statistically analyzed. aPDT as an adjunct to SRP did not demonstrate statistically significant advantages on clinical parameters when compared with SRP alone. In general no statistic significant differences between groups were observed (p &lt0.05). Levels of anti-inflammatory cytokines and bacterial species were comparable in both groups at day 90 after treatment. Periodontal treatment with SRP + aPDT in multiples episodes was not able to improve results in the non-surgical treatment of chronic periodontitis in smokers when compared SRP alone, without the use of systemic antibiotics

Orientador: Celso Valentim Santilli / Resumo: A incidência de infecções cutâneas causadas por agentes bacterianos resistentes ao tratamento convencional com antibióticos tornou-se um grande problema de saúde pública. Algumas abordagens terapêuticas alternativas têm sido propostas, dentre elas a terapia fotodinâmica antimicrobiana (aPDT) e hipertermia magnética (HM). A aPDT envolve a geração de espécies reativas de oxigênio (EROs) por meio da combinação de luz, oxigênio molecular e um agente fotossensibilizador (FS), e a HM promove o aumento da temperatura de uma região anatômica submetendo nanopartículas superparamagnéticas de óxido de ferro (SPIONPs) a um campo magnético alternado (CMA). O FS de escolha para este estudo foi a curcumina (CUR), muito usado em aPDT, porém tem a fotoavidade fortemente afetada em meios aquosos devido à tendência a agregação. Visando evitar isto, além de promover a melhoria da administração da CUR e a combinação de aPDT e HM num único material foi proposto neste trabalho a síntese de conjugados SPIONP-curcumina (Magh-CUR), assim como a sua avaliação em aPDT e HM contra Staphylococcus aureus em fase planctônica. Este material magnético foi preparado por uma metodologia de coprecipitação alcalina seguida por uma etapa de conjugação com a CUR. Os conjugados foram caracterizados por difração de raios X, microscopia eletrônica de transmissão, potencial zeta, espectroscopia no infravermelho com transformada de Fourier, termogravimetria, magnetometria e ensaios de HM. Os resultados evidenciaram que ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The incidence of cutaneous infections caused by bacteria resistant to conventional antibiotic treatment has become a major public health problem. Some alternative therapeutic approaches have been proposed, among them antimicrobial photodynamic therapy (aPDT) and magnetic hyperthermia (MH). The aPDT involves the generation of reactive oxygen species (ROS) by using the combination of light, molecular oxygen and a photosensitizing agent (PS), and MH promotes temperature increase of an anatomic region by subjecting superparamagnetic iron oxide nanoparticles (SPIONPs) to an alternating magnetic field (AMF). The PS of choice for this study was curcumin (CUR), widely used in aPDT, but still has its photoactivity strongly affected in aqueous media due to the tendency of aggregation. In order to solve this problem and to promote the improvement of the administration of CUR, we propose the combination of CUR and SPIONPs of maghemite in a single material. Therefore, the objective of this work was to synthesize a SPIONPs-curcumin conjugate (Magh-CUR) and, mediated by the aPDT, to evaluate it against Staphylococcus aureus in the planktonic phase. This magnetic material was prepared by an alkaline coprecipitation method followed by a conjugation step with CUR. The conjugates were characterized by X-ray diffraction, electron transmission microscopy, zeta potential, infrared spectroscopy with Fourier transform, thermogravimetry, magnetometry, MH assays. The characterizations showed that the ... (Complete abstract click electronic access below) / Mestre

A técnica do enxerto de granulação óssea tem demonstrado bons resultados na recuperação do periodonto e na melhora dos parâmetros clínicos dos dentes com comprometimento periodontal. Pouco se sabe porém, a respeito de qual tipo de tratamento de superfície radicular se faz mais condizente com o emprego dessa técnica. O objetivo dessa pesquisa foi avaliar a proliferação de células de granulação óssea sobre fragmentos radiculares com os seguintes tratamentos de superfície: Controle - somente raspagem, EDTA, terapia fotodinâmica (PDT- laser InGaAIP - 30mW, 30s, 45J/cm², 660nm + azul de toluidina), e ácido cítrico com tetraciclina. Todos os grupos teste receberam previamente tratamento com raspagem e alisamento com 20 golpes de cureta. Células de granulação óssea foram cultivadas em quadruplicata sobre os fragmentos por um período de 24, 48 e 72 horas. Após esse período de cultivo os fragmentos foram fixados para análise em microscópio eletrônico de varredura (MEV). Cinco campos por fragmento foram usados para a visualização e contagem de células aderidas a superfície radicular (centro, campo superior direito e esquerdo e campo inferior direito e esquerdo). A análise da calibração do examinador foi feita através de uma combinação de testes estatísticos como erro casual de Dahlberg, erro sistemático e correlação de Pearson (p<0,05). A análise da amostra foi realizada através do ANOVA de medidas repetidas complementado por Tukey, com nível de significância de 5% (p<0,05). Os resultados demostraram diferenças estatisticamente significantes, quanto ao numero de células, para as superfícies tratadas com terapia fotodinâmica no período de 72 h (p<0,05). Através de nossos resultados concluímos que o tratamento radicular com terapia fotodinâmica favorece a proliferação de células de granulação óssea humanas in vitro. / The osseous granulation graft has been demonstrating good results on the periodontal healing, resulting the improvement of clinical periodontal parameters. There are very few knowledge about what kind of dental surface would be more proper for the application of this technique. The aim of this study was to evaluate the proliferation of osseous granulation cells on human root fragments treated by different techniques as scaling and root planning (control), citric acid plus tetracycline, EDTA and photodynamic therapy (PDT InGaAIP, 45J/cm², 30mW, 30s, 660nm, toluidine blue O). All test groups were previously treated which 20 curette strikes. Osseous granulation cells was culture in quadruplicate on these fragments for 24h, 48h and 72h. After that, all fragments were fixed and prepared for analysis in Scanning Electron Microscopy (SEM). Aiming to counting the cells adhered on the roots, we obtained electron micrographs of 5 areas (center, upper right and left field, lower right and left field). The examiner calibration was analyzed by Dahlberg Casual Error measurement, systematic error test and Pearson correlation test (p<0.05). Statistical analysis was performed by ANOVA, followed by Tukey test, with a 5% level of significance (p<0.05). There were significant differences in cell number after 72h culture in favor of PDT group (p<0,05). We can conclude that the surface treatment of roots which PDT favor the proliferation of osseous granulation cells in vitro.

A candidose bucal acomete grande número de pessoas mundialmente, sendo associada a condições como imunossupressão, radioterapia, tabagismo, higiene bucal, idade, xerostomia e uso de próteses removíveis. Uma importante abordagem terapêutica para essas infecções consiste nos medicamentos antifúngicos, que podem apresentar efeitos colaterais e resultar na resistência dos patógenos. Nesse contexto, a terapia fotodinâmica (PDT) faz-se interessante como alternativa capaz de minimizar essas limitações. Protocolos de PDT poderão ser mais facilmente assimilados à prática clínica se empregarem materiais já aprovados para uso odontológico. Sendo assim, este estudo propõe avaliar o uso de PDT com eritrosina como fotossensibilizador, irradiada por um LED azul, usando-se um modelo animal. Quarenta camundongos tiveram candidose induzida sobre o dorso da língua por meio de imunossupressão e inoculação com Candida albicans. Após estabelecimento da lesão durante cinco dias, os animais receberam um dentre quatro tratamentos possíveis: aplicação da eritrosina 5% seguida de irradiação pelo LED (E+L+); aplicação de eritrosina, somente (E+L-); salina seguida pela irradiação (EL+); e somente salina (E-L-). A fim de distinguir possíveis efeitos colaterais, os mesmos tratamentos foram aplicados em 12 camundongos sem a indução de candidose. O número de unidades formadoras de colônia de C. albicans foi contado após o tratamento, e a mucosa foi submetida à análise histológica para determinar o grau de inflamação. Os dados dos grupos foram comparados por meio de ANOVA e teste de Kruskal-Wallis, (&alpha;=0,05). Não foram detectadas diferenças significativas entre os grupos testados quanto à contagem de colônias de C. albicans e o grau de inflamação. O infiltrado inflamatório foi classificado como discreto na maioria dos casos. Os animais com candidose bucal induzida e os saudáveis que foram submetidos aplicação de eritrosina 5% e irradiação pelo LED apresentaram infiltrado inflamatório mais acentuado e lesões mais evidentes na camada epitelial, sugerindo que este protocolo ocasionou danos aos tecidos orais. / Oral candidosis affects many people worldwide and is associated with conditions such as immunosuppression, radiation, smoking, oral hygiene, age, xerostomia and use of removable prostheses. An important therapeutic approach for these infections consists of antifungal drugs, which have side effects and can result in the resistance of pathogens. In this context, photodynamic therapy (PDT) is an interesting alternative able to overcome those limitations. PDT protocols can be more easily assimilated in clinical practice if they employ materials already approved for use in dentistry. Thus, this study evaluate the PDT with the use of erythrosine as a photosensitizer, irradiated by a blue LED light, using an animal model. Forty mice had candidosis induced on the tongue by the immunosuppression and inoculation with Candida albicans. After five days that the lesion is established, the animals received one of four possible treatments: application of 5% erythrosine and irradiation by the LED (E+L+); erythrosine application, only (E+L- ); saline followed by irradiation (E-L+); and only saline (E-L-). In order to distinguish potential side effects, the same treatments were applied in 12 mice without induced candidosis. Colony-forming units (CFU) of C. albicans were counted after treatment, and the mucosa was subjected to histological analysis to determine the degree of inflammation. The data of the groups were compared using ANOVA and Kruskal-Wallis test (&alpha;=0.05). No significant difference was detected among the groups tested for the number of CFU of C. albicans and the degree of inflammation. The inflammatory infiltrate was classified as mild in most cases. Animals with induced oral candidosis and the healthy ones exhibited more pronounced inflammatory infiltrate and lesions in the epithelial layer following PDT by 5% erythrosine and irradiation by the LED light. Such findings suggest that this protocol resulted in damage to oral tissues.