Dissertations / Theses on the topic 'Toluidine blue'

Formulations of Methylene Blue (MB) and toluidine blue (TB) with ethanol or oxygen carrier were evaluated for antimicrobial effect on Pseudomonas aeruginosa biofilms. Formulations photoactivated had photosensitizers (Ps) (250 μM) diluted in PBS (pH 5.6 - sodium acetate / acetic acid), 10% ethanol (buffer:ethanol 90:10), 20% ethanol (buffer:ethanol 80:20) and perfluordecalin emulsion, buffer and triton-X100 (60: 35: 5). The controls were biofilms untreated and without photoactivation. The production of singlet oxygen was measured by photo-oxidation of 1,3-difenilisobenzofuran.The data of photo-oxidation and CFU (log10) were evaluated by One-Way ANOVA and post hoc Tukey and Dunnett, respectively. Student t-test assessed differences between the Ps in the same formulation. MB showed higher singlet-oxygen production. Buffer or etanol 10% hadn´t antimicrobial effect. Ethanol 20% reduced the bacterial viability (1.2 log10, p = 0.009). The lower mean CFU were observed with MB and ethanol 10% (P = 0.052; reduction of 2.18 log10) and 20% (P = 0.053; reduction of 2.18 log10). Microbial reductions in water MB and TB were 1.13 log10 and 1.10 log10, respectively. With carredor reductions were 1.74 and 1.70 for MB and TB, respectively. MB formulations containing ethanol are comparable to some antibiotics in treating P. aeruginosa biofilms in vitro.<br>Formulações de Azul de Metileno (AM) e Azul de Toluidina (AT) contendo etanol ou carreador de oxigênio foram avaliadas quanto ao efeito antimicrobiano em biofilmes de Pseudomonas aeruginosa. Formulações fotoativadas continham os fotossensibilizados (Fs) (250 μM) diluídos em água tamponada (pH 5,6 - acetato de sódio/ácido acético), etanol 10% (tampão:álcool etílico 90:10), etanol 20% (tampão:álcool etílico 80:20) e emulsão com perfluordecaleno, tampão e triton-X100 (60:35:5). Biofilmes sem tratamento e expostos ao tampão, etanol 10% e 20% sem fotoativação foram avaliados como controles. A produção de oxigênio singleto dos Fs foi mensurada por meio da foto-oxidação do 1,3-difenilisobenzofuran. Os dados de foto-oxidação e UFC (log10) foram avaliados por One-Way ANOVA e post hoc Tukey e Dunnett, respectivamente. Teste t-Student avaliou diferenças entre os Fs na mesma formulação. AM apresentou maior produção do oxigênio-singleto. Tampão ou etanol 10% não tiveram efeito antimicrobiano. Etanol 20% reduziu signficativamente a viabilidade bacteriana (1,2 log10, P=0,009). As menores médias de UFC log10 foram observadas com AM e etanol 10% (P=0,052; redução de 2,18 log10) e 20% (P=0,053; redução de 2,18 log10). Reduções microbianas do AM e AT em água foram de 1,13 log10 e 1,10 log10, respectivamente. Com o carredor as reduções foram de 1,74 e 1,70 para AM e AT, respectivamente. Formulações de AM contendo etanol são comparáveis a alguns antibióticos no tratamento de biofilmes de P. aeruginosa in vitro.

A peri-implantite acomete um número crescente de indivíduos e os protocolos de tratamento objetivam descontaminar as superfícies dos implantes e torná-las novamente osseointegráveis. Porém, ainda não foi estabelecido um padrão ouro de tratamento para peri-implantite. Este estudo testa o poder de descontaminação do laser em baixa intensidade (LBI), da terapia fotodinâmica (PDT) e do azul de toluidina O (TBO). Sobre discos de titânio de 1,5 mm de espessura e 4,0 mm de diâmetro de superfícies lisas ou rugosas foi permitida a deposição natural de biofilme microbiano por 7 dias após os quais os discos foram divididos em grupos experimentais (15 discos por grupo), de acordo com o método de descontaminação: grupo LBI (laser de InGaAlP de 660 nm, 30 mW, 45 J/cm2 no modo continuo por 30 s); grupo PDT; grupo TBO (aplicação de TBO por 60 s); grupo controle positivo (C) sem tratamento e grupo controle negativo (NC) estéril. Os discos foram implantados em tecido conjuntivo subcutâneo de ratos e, após 7, 28 e 84 dias, foram obtidas 5 biópsias de cada grupo para análise em microscopia óptica. Implantes lisos e rugosos não diferiram com relação ao grau de fibrosamento e severidade do infiltrado inflamatório, mas a área do tecido reacional perimaterial foi maior nos rugosos (2,6 ± 3,7 x 106 µm2) do que nos lisos (1,9 ± 2,6 x 106 µm2). O grupo C foi o que apresentou menor grau de fibrosamento do tecido reacional (1), mas somente houve diferença estatisticamente significante (p = 0,0230) entre os grupos C e NC. A severidade do infiltrado inflamatório somente diferiu significantemente entre os grupos aos 7 dias, quando o grupo NC apresentou o menor escore (2) em comparação aos demais grupos (3). Com relação à área do tecido reacional perimaterial, só houve diferenças entre os grupos aos 7 dias, quando o grupo C apresentou maior área que os demais grupos (9,11 ± 2,10 x 106 µm2), porém, sem diferença estatisticamente significante em comparação aos grupos LBI e TBO. A área apresentada pelo grupo PDT (4,34 ± 1,49 x 106 µm2) só foi inferior ao grupo NC (0,65 ± 0,23 x 106 µm2). A espessura do tecido reacional não confirmou a diferença entre os discos de superfície lisa e os de superfície rugosa (p = 0,2801), mas confirmou todas as demais comparações relativas à área, sendo que o grupo NC apresentou menor espessura que os demais somente aos 7 dias. O grupo NC apresentou menor espessura de tecido reacional do que os demais grupos, mas essa diferença foi estatisticamente significante somente aos 7 dias. Os demais grupos não diferiram entre si quanto a esse parâmetro. Concluiu-se que implantes rugosos ou lisos contaminados por biofilme microbiano e tratados com LBI, PDT e TBO não diferem entre si quanto aos parâmetros inflamatórios teciduais, parecendo haver leve superioridade da PDT sobre os demais tratamentos apenas no período mais precoce de observação. Porém, com o passar do tempo, todos os tratamentos se equiparam a implantes estéreis quanto à reação tecidual perimaterial.<br>The peri-implantitis is a disease that is increasing in a number of individuals and the protocols of treatment has intended to decontaminate the implant surfaces and makes this biocompatible again. However, until this moment, a gold standard for peri-implantitis treatment was not established. The present study tests the power of decontamination of low intensity laser (LBI), photodynamic therapy (PDT) and toluidine blue O (TBO). The natural deposition of microbian biofilm was allowed on titanium discs with 1,5 mm of thickness and 4,0 mm of diameter and smooth and grooved surfaces during 7 days when the discs were separated in experimental groups (15 discs each group), in accordance with the decontamination method: LBI group (decontamination with InGaAlP laser, 660 nm, 30 mW, 45 J/cm2 in continuous way during 30 s); PDT group; TBO group (application of TBO during 60 s); controlled positive group (C) without any kind of treatment and controlled negative group (NC), sterile. The discs had been implanted in rats subcutaneous connective tissue and, after 7, 28 and 84 days, had been gotten 5 biopsies from each group for histology processing. Smooth and grooved implants did not differ on fibrosis grade and severity of the inflammatory infiltrate, however the perimaterial reaction tissue area was bigger on grooved implants (2,6 ± 3,7 x 106 µm2) that on smooth implants (1,9 ± 2,6 x 106 µm2). The C group was the one that showed the smallest reaction tissue fibrosis grade (1), but only was significant statistical difference (p = 0,0230) between C and NC groups. The severity of inflammatory infiltrate only significant differed between the groups on 7 days, when the NC group showed the smallest score (2) in comparison to the others groups (3). In relation to the perimaterial reaction tissue area, only were differences between the groups on 7 days, when C group showed bigger area than the others (9,11 ± 2,10 x 106 µm2), nevertheless, without significant statistical difference in Comparison to LBI and TBO groups. The area presented by PDT group (4,34 ± 1,49 x 106 µm2) only was inferior to NC group (0,65 ± 0,23 x 106 µm2). The reaction tissue thickness did not confirmed the difference between smooth and grooved surfaces discs (p = 0,2801), however it confirmed all the comparisons related to the area parameter, where NC group showed smaller thickness than the others only on 7 days. NC group showed smaller thickness than the others, but this difference was statistical significant only on 7 days. The other groups did not differ among them in this parameter. It was concluded that grooved and smooth implants contaminated by a microbian biofilm ant treated with LBI, PDT and TBO did not differed among them in relation to tissue inflammatory parameters, seems that is a light superiority of PDT in relation to the others only in the earliest period of observation. Nevertheless, with the time, all the treatments were equally to sterile implants in relation to perimaterial reaction tissue.

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.<br>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.

Made available in DSpace on 2014-10-09T12:46:02Z (GMT). No. of bitstreams: 0<br>Made available in DSpace on 2014-10-09T14:04:58Z (GMT). No. of bitstreams: 1 07496.pdf: 4298184 bytes, checksum: 0ea1a09cef64fb7f7cb7c36f765c879b (MD5)<br>Dissertacao (Mestrado Profissionalizante Lasers em Odontologia))<br>IPEN/D-MPLO<br>Intituto de Pesquisas Energeticas e Nucleares, IPEN/CNEN-SP; Faculdade de Odontologia, Universidade de Sao Paulo

38 PIVs carried through in two laboratories of the Uberaba MGBRASIL had been used in this work with the objective to evaluate the relationship between condensation chromatin and head morphometry of spermatozoa and cleavages and blastocyst formation rates using semen of zebu bulls with normal routine tests of fertility. Smears of semen stained with of Toluidine Blue and computer image analysis had been used to evaluate the morphometry and chromatin condensation. Area (A), perimeter (P), width (L), length (C), width:length ratio (L/C), ellipticity (E), shape factor (FF), side symmetry (SL), anterior-posterior symmetry (SAP) and the three first Fourier values (F0, F1, F2) had been evaluated in the morphometric analysis. The chromatin was evaluated in relation to intensity of condensation and homogeneity. The results allowed concluding that the influence of light variations in the sperm head morphometry and the chromatin condensation on the cleavage and number of normal morfologic blastocyst in the PIV is small.<br>Foram utilizados os dados de 38 programações de produção in vitro de embriões (PIV), realizadas em dois laboratórios da cidade de Uberaba-MG-Brasil, com o objetivo de se avaliar a correlação entre compactação da cromatina e morfometria da cabeça de espermatozóides com a taxa de clivagem e formação de blastocisto utilizando sêmen de touros zebuínos com testes de rotina normais para fertilidade. Foram utilizados esfregaços de sêmen corados com Azul de Toluidina e análise de imagem por computador para avaliar a morfometria e a compactação de cromatina. Na análise morfométrica foram avaliados: área (A), perímetro (P), largura (L), comprimento (C), razão largura:comprimento (L/C), elipsidade (E), fator forma (FF), simetria lateral (SL), simetria ântero-posterior (SAP) e descritores Fourier com amplitude de 0 a 2 (F0, F1, F2). A cromatina foi avaliada quanto à intensidade de compactação e homogeneidade. Os resultados permitiram concluir que é discreta a influência de pequenas variações observadas na morfometria da cabeça e na compactação da cromatina espermática sobre a clivagem e número de blastocistos morfologicamente normais na PIV.<br>Mestre em Ciências Veterinárias

Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq<br>Babesiosis is a hemolytic tick-borne disease caused by intraerythrocytic protozoal parasites of the genus Babesia. This is a disease of high incidence in the South region of Brazil and responsible for considerable economic losses. Clinical-pathological diagnosis can be made by demonstrating the parasite by examining blood smears or fresh tissues. However, frequently, only formalin-fixed organs are sent to pathology laboratories, which makes it impossible to perform the smears. The main objective of this study was to find alternative histochemical techniques capable to improve the identification of intraerythrocytic B. bovis in histological sections. For this study, tissue samples from 50 bovine necropsy cases were retrospectively analyzed, which had been fixed in formalin and routinely processed for histopathology. The cases were divided into a control group, consisting of 12 cases of cerebral babesiosis with characteristic pink-cherry gray matter (group A), and 38 cases suggestive of cattle tick fever (group B), according to necropsy protocols. Histochemical techniques of Alcian Blue, Methylene Blue, Toluidine Blue, Giemsa, Gram (McCallum-Goodpasture method), Grocott, Periodic Acid of Schiff and Ziehl-Neelsen were tested. Among these, it was observed that the techniques of Methylene Blue and Toluidine Blue allowed to observe morphological and dye characteristics in a clearer way, aiding in the identification of B. bovis. In addition, several clinical-epidemiological and anatomopathological parameters of babesiosis caused by B. bovis were established.<br>A babesiose é uma doença hemolítica transmitida por carrapatos e causada por protozoários intraeritrocitários do gênero Babesia. Esta é uma doença de incidência elevada na Região Sul do Brasil e responsável por perdas econômicas consideráveis. O diagnóstico clínico-patológico pode ser feito através da demonstração do parasita pelo exame de esfregaços sanguíneos ou de tecidos frescos. Entretanto, com frequência, somente órgãos fixados em formol são remetidos para laboratórios de patologia, o que impossibilita a realização do esfregaço. O principal objetivo deste estudo foi encontrar técnicas histoquímicas alternativas e capazes de aprimorar a evidenciação de B. bovis intraeritrocitária. Para este estudo, foram analisadas, retrospectivamente, amostras de tecidos de 50 casos de necropsias de bovinos, as quais haviam sido fixadas em formol e processadas rotineiramente para histopatologia. Os casos foram divididos em um grupo controle, constituído por 12 casos de babesiose cerebral com substância cinzenta encefálica róseo-cereja característica (grupo A), e 38 casos sugestivos de tristeza parasitária bovina (grupo B), conforme os protocolos de necropsias. Foram testadas as técnicas histoquímicas de Azul Alciano, Azul de Metileno, Azul de Toluidina, Giemsa, Gram (método de McCallum-Goodpasture), Grocott, Ácido Periódico de Schiff e Ziehl-Neelsen. Dentre estas, observou-se que as técnicas de Azul de Metileno e Azul de Toluidina permitiram observar características morfológicas e tintoriais de maneira mais nítida, auxiliando na identificação de B. bovis. Adicionalmente, foram estabelecidos vários parâmetros clínico-epidemiológicos e anatomopatológicos da babesiose por B. bovis.

Made available in DSpace on 2014-12-17T15:30:59Z (GMT). No. of bitstreams: 1 EmanuelleDVD_DISSERT.pdf: 1398010 bytes, checksum: 85e1d1f82b33da3e697eacbf6655c267 (MD5) Previous issue date: 2011-08-26<br>Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior<br>Photodynamic therapy (PDT) has been proposed as an alternative method for the treatment of biofilm-dependent oral diseases like dental caries. This therapy consists of simultaneous action of a visible light (L) and a photosensitizer (FS) in the presence of oxygen, which leads to production of different reactive oxygen species that can interact with the bacterial cell components, and promote cell death. This study aims to evaluate the antimicrobial action of PDT on oral bacteria in suspension, as well as the formation of mono and multi-species biofilms, in vitro, from a standard strain of Streptococcus mutans (ATCC 25175) and saliva samples, respectively. The dye methylene blue (MB) and toluidine blue (TB) were used at a concentration of 100 mg/ L and activated by halogen light (600 to 750 nm) from a modified hand held photopolymerizer (Ultralux ?, Dabi Atlante, Ribeir?o Preto , S?o Paulo, Brazil.). Planktonic cultures were prepared and submitted to different experimental conditions: 1. PDT using TB 2. PDT using MB, 3. L+ FS- , 4. TB + L - ; 5. MB+ L-; 6. L- FS- (negative control) and 7. administration of 0.12% chlorhexidine digluconate (positive control) (Periogard ?, Colgate-Palmolive Company, New York, USA). The immediate and mediated action of PDT on bacterial suspensions, as well as its effect on biofilm formation were observed from the number of colony-forming units per milliliter (CFU/mL) and measures optical density (OD). The data were statistically analyzed using the Kruskal-Wallis and Mann-Whitney test for the significance level of 5%. According to the results, the PDT showed no antibacterial action on suspensions of S. mutans, regardless of the dye used. PDT with MB activated by halogen light was able to reduce 86.6% CFU/mL multi-species planktonic cultures, however, this reduction was not significant (p > 0.05). PDT showed antibacterial effect, mediate on multi-species planktonic cultures with TB (p < 0.001) and MB (p < 0.001), activated by halogen light. PDT was able to prevent the formation of multispecies biofilm, through the activation of TB by halogen light (p = 0.01). We conclude that activation of the dye toluidine blue and methylene blue, by halogen light (PDT) showed antimicrobial activity, compared to multi-species planktonic cultures prepared from saliva samples<br>A terapia fotodin?mica (TFD) tem sido proposta como um m?todo alternativo para o tratamento de doen?as orais biofilme-dependentes, como a c?rie dent?ria. Tal terapia consiste na a??o simult?nea de uma fonte luminosa (FL) e de um agente fotossensibilizante (FS), na presen?a de oxig?nio tecidual, levando ? produ??o de radicais livres que ao interagirem com componentes celulares bacterianos, promovem a morte celular. Este estudo tem como objetivo avaliar a a??o antimicrobiana da TFD sobre bact?rias orais em suspens?o, bem como na forma??o de biofilmes mono e multiesp?cies, in vitro, a partir de uma cepa padr?o de Streptococcus mutans (ATCC 25175) e de amostras de saliva, respectivamente. Os corantes azul de metileno (CM) e azul de toluidina (CT) foram administrados na concentra??o de 100 mg/L e ativados por luz hal?gena (600 a 750 nm) proveniente de um aparelho fotopolimerizador modificado (Ultralux?, Dabi Atlante, Ribeir?o Preto, S?o Paulo, Brasil.). Suspens?es monoesp?cies e mistas foram preparadas e submetidas a diferentes condi??es experimentais: 1. TFD na presen?a de CT; 2. TFD na presen?a de CM; 3. irradia??o sem administra??o de qualquer corante; 4. sensibiliza??o com CT e aus?ncia de luz; 5. sensibiliza??o com CM e aus?ncia de luz; 6. aus?ncia de irradia??o e sem qualquer corante (controle negativo) e 7. administra??o de solu??o de digluconato de clorexidina a 0,12% (controle positivo) (Periogard?, Colgate-Palmolive Company, Nova York, EUA). A a??o imediata e mediata da TFD sobre as suspens?es bacterianas, bem como seu efeito sobre a forma??o de biofilme foram verificados, a partir do n?mero de unidade formadoras de col?nias por mL (UFC/mL) e de medidas de densidade ?tica (DO). Os dados obtidos foram analisados estatisticamente atrav?s dos Testes Kruskal-Wallis e Mann-Whitney, para o n?vel de signific?ncia de 5%. De acordo com os resultados, a TFD n?o apresentou a??o antibacteriana sobre as suspens?es de S. mutans, independente do corante utilizado. A TFD com CM ativado por luz hal?gena foi capaz de reduzir em 86,6% o n?mero de UFC/mL diante de suspens?es multiesp?cies, entretanto, tal redu??o n?o foi significativa (p > 0,05). A TFD apresentou efeito antibacteriano, mediato, sobre suspens?es mistas, quer na presen?a de CT (p < 0,001) ou CM (p < 0,001), ativados por luz hal?gena. A TFD preveniu a forma??o de biofilme multiesp?cie, atrav?s da ativa??o de CT por luz hal?gena (p = 0,01). Conclui-se que a ativa??o dos corantes azul de toluidina e azul de metileno, pela luz hal?gena (TFD) apresentou a??o antimicrobiana, frente a suspens?es multiesp?cies preparadas a partir de amostras de saliva, ressaltando a possibilidade da utiliza??o da terapia fotodin?mica como um m?todo coadjuvante no tratamento de les?es de c?rie dent?ria

O aumento significativo de micoses decorrentes do crescimento do número de indivíduos imunocomprometidos, da emergência de novas espécies de fungos patogênicos e do surgimento de patógenos resistentes aos antifúngicos atualmente utilizados é um sério problema de saúde pública. Nesse contexto, é extremamente desejável o desenvolvimento de novas técnicas para o controle de fungos. A TFD (terapia fotodinâmica), inicialmente desenvolvida como uma alternativa terapêutica para o câncer, é um processo que envolve a administração de um fotossensibilizador que se acumula preferencialmente nas células-alvo e que pode ser ativado por exposições à luz. A ativação do fotossensibilizador leva à formação de espécies reativas de oxigênio que são capazes de danificar lipídios, proteínas e ácidos nucléicos, provocando a morte da célula. A TFDA (terapia fotodinâmica antimicrobiana) pode ser utilizada tanto para o controle de micoses localizadas como para a eliminação de espécies patogênicas de fungos do ambiente. Neste estudo, foi avaliada a atividade fotossensibilizadora e foram testados parâmetros funcionais como concentração do fotossensibilizador (1 a 400 g mL-1); tempo de pré-incubação com o fotossensibilizador (0 a 60 min.); doses (90 e 180 kJ m-2) e intensidade de luz visível (50 W m-2) para a fotossensibilização de: (1) fotossensibilizadores comerciais atualmente utilizados na TFDA, como o azul de metileno (MB) e o azul de ortotoluidina (TBO), e (2) complexos nitrosilos de rutênio. Os experimentos foram conduzidos com conídios dos fungos-modelo Metarhizium anisopliae e Aspergillus nidulans. Tanto o MB como o TBO, na presença da luz, foram capazes de inativar os conídios das duas espécies de fungos. A inativação foi próxima de 100%, para ambas as espécies, quando a combinação apropriada (concentração do fotossensibilizador e tempo de exposição à luz) foi utilizada. Nenhum dos dois corantes, na ausência da luz, inativou os conídios das duas espécies. O tempo de pré-incubação com os fotossensibilizadores (MB e TBO) não foi um parâmetro determinante para a eficiência da fotoinativação dos conídios. De maneira geral, a inativação dos conídios foi maior nas fotossensibilizações com MB e TBO por 60 min do que nas por 30 min. Os conídios verdes e amarelos foram mais tolerantes à fotossensibilização com MB e TBO do que os conídios brancos e violetas, indicando que a pigmentação dos conídios influencia na fotoinativação. O nitrosilo de rutênio [Ru(NH.NHq)(tpy)NO](PF6)3 não foi capaz de inativar os conídios de nenhuma das espécies, em nenhuma das condições avaliadas. A fotoinativação dos conídios de M. anisopliae com MB e TBO não ocorreu quando a fotossensibilização foi conduzida em meio de cultura líquido PDB.<br>The significative increase of mycoses resulting from the growing number of immunocompromised individuals, from the emerging of new species of pathogenic fungi, and from the emerging of pathogens resistant to the antimycotics used nowadays is a serious public health problem. In such scenario, the development of new fungal control techniques is highly desirable. Initially developed as a therapeutical alternative for cancer, PDT (photodynamic therapy) is a process that involves the use of a photosensitizer that preferrably accumulates in the target-cells and that can be light-activated. The activation of the photosensitizer leads to the generation of reactive oxygen species that damage lypids, proteins, and DNA, and kills the cell. APDT (antimicrobial photodynamic therapy) can be used to control localized infections as well as to kill pathogenic fungi in the environment. In this study, we evaluated the photoinactivation by MB (methylene blue), TBO (toluidine blue), and nitrosyl ruthenium complexes in Metarhizium anisopliae and Aspergillus nidulans conidia. We also tested parameters such as photosensitizer concentration (1 to 400 g mL-1), pré-incubation time with the photosensitizer (0 to 60 min.), light doses (90 and 180 kJ m-2), and visible light irradiance (50 W m-2). Both MB and TBO photoinactivated the conidia of the two species of fungi. Inactivation was close to 100% for both species when the appropriate combination between photosensitizer concentration and light exposure time was used. None of the two dyes in the dark inactivated the conidia of the two species. Pre-incubation time with the photosensitizers (MB and TBO) was not a determinant parameter for photoinactivation efficiency. Conidia inactivation was higher in photosensibilizations with MB and TBO for 60 min than in those for 30 min. Green and yellow conidia were more tolerant to photosensibilization than mutants with white and violet conidia, indicating that conidia pigmentation influences photosensibilization. Nitrosyl ruthenium [Ru(NH.NHq)(tpy)NO](PF6)3 was not capable to photoinactivate conidia of any of the species, in any of the evaluated conditions. M. anisopliae conidia photoinactivation with MB and TBO did not occur when photosensibilization was conducted in PDB media.

Diabetic ketoacidosis (DKA) is a life-threatening condition that can appear in patients with diabetes. High ketones in the blood lead to acidity of the blood. For DKA diagnosis and management, ketones such as hydroxybutyrate (HB) can be used to quantify the severity of the disease. The fabrication of electrochemical biosensors for the detection of HB is attractive since their capability to deliver fast response, high sensitivity, good selectivity and potential for miniaturisation. In this thesis, an integrated electrode system was prepared for the detection of HB. Laser-induced graphene (LIG) with a 3D porous structure was used as the flexible platform. Poly (toluidine blue O) (PTB) was electro-deposited on LIG (PTB/LIG) under the optimised conduction (pH of 9.7 and from 0.4 to an upper cyclic potential of 0.8 V). The single PTB/LIG working electrode demonstrated excellent performance towards the detection of NADH with a linear range of 6.7 M to 3 mM using chronoamperometry, high sensitivity of detecting NADH and excellent anti-fouling ability (94 % response current retained after 1500 s). Further integration of the 3-electrode system realised the static amperometric detection of NADH over the range of 78 M to 10 mM. Based on the excellent performance of PTB/LIG to NADH sensing, hydroxybutyrate dehydrogenase was immobilised via encapsulation with chitosan and polyvinyl butyral (PVB) which was used for HB biosensing over the linear range of 0.5 M to 1 mM with NAD+ dissolved in solution. In addition, the co-immobilisation of NAD+ and HBD on PTB/LIG was conducted by optimisation of enzyme and NAD+ amount per electrode, which shows excellent reproducibility and satisfactory HB biosensing performance. Further experiments to improve the long-term stability of the enzyme electrode is expected in the future. The proposed integrated electrode system also possesses the potential to extend to a multichannel sensor array for the detection of multiple biomarkers (e.g. pH and glucose) for diagnosis and management of DKA.

O câncer de mucosa oral é um problema de saúde pública, com maior incidência em homens acima de 50 anos. Uma das manifestações clínicas mais precoces do câncer da mucosa oral são as lesões leucoqueratósica. O aspecto clínico não homogêneo e o tamanho maior de 200mm2, em mucosa não queratinizada como a do assoalho da boca e ventre da língua, são aspectos que implicam na possibilidade de evolução da lesão. A dificuldade do diagnóstico das lesões precoces está na seleção do local a ser biopsiado principalmente frente a lesões extensas e heterogêneas. O objetivo desta pesquisa é avaliar se a utilização do exame clínico juntamente com os métodos auxiliares de diagnóstico das lesões leucoqueratósica da mucosa oral (VELscope®, Azul de toluidina e Solução de lugol) contribui para uma maior precisão do diagnóstico de displasias nestas lesões quando comparado ao histopatológico. E, identificar os padrões morfológicos destas lesões quando avaliadas por meio da microscopia confocal reflectante. Foram selecionados 30 pacientes, maiores de 18 anos, portadores de lesão clínica compatível com leucoplasia oral triados no ambulatório da disciplina de Estomatologia Clínica da Faculdade de Odontologia da Universidade de São Paulo e que precisavam ser submetidos à biópsia para o estabelecimento do diagnóstico final. Foram realizados os testes de Azul de Toluidina, Solução de Lugol, VELscope®, microscopia confocal reflectante e posteriormente, a biópsia incisional para obtenção do diagnóstico final. Os pacientes incluídos possuíam média de idade 60,66 anos, sendo 70% (21/30) do gênero feminino e 30% (9/30) do gênero masculino. O tabagismo foi relatado por 16,7% (5/30) dos pacientes, sendo 60% (3/5) homens. A associação do tabagismo e etilismo foi relatada em 10% (3/30) dos pacientes, o tabagismo isoladamente por 6,6% (2/30) e o de etilismo por 3,3%(1/30). A associação dos hábitos e o etilismo isolado foram relatados somente por homens. Enquanto que o hábito de tabagismo isolado foi relatado somente por mulheres. Com o uso do VELscope® todas as lesões apresentaram perda da fluorescência tecidual sendo, portanto todas positivas para este método. Com a Solução de Lugol, 100% das lesões não coraram, sendo portanto positivas para este método. O exame histopatológico permitiu o diagnóstico de 30% (9/30) casos de carcinoma epidermoide, 10% (3/30) de neoplasia intraepitelial, 20% (6/30) de líquen plano oral e 40% (12/30) de líquen simple crônico oral. Para o uso do VELscope® e do Lugol foi observada uma sensibilidade de 100% e uma especificidade de 50%.Para o Azul de Toluidina foi observada uma sensibilidade de 63,15%- 66,66% e especificidade de 75,67%-82,35%.Na Microscopia Confocal Reflectante foram definidos padrões para os quatro diferentes diagnósticos observados. Os métodos auxiliares de diagnóstico por cromoscopia e fluorescência de mucosa se mostraram eficientes no auxílio da delimitação da extensão da lesão e a Microscopia Confocal Reflectante permitiu a visualização de padrões morfológicos das lesões brancas de forma não invasiva. A utilização da Microscopia Confocal Reflectante permitiu também a determinação de padrões morfológicos para o carcinoma epidermoide, a neoplasia intraepitelial, o líquen plano oral e o líquen simples crônico oral e se mostrou eficiente quando comparada ao exame histopatológico.<br>Cancer of the oral mucosa is a public health problem, with higher incidence in men above 50 years. One of the earliest manifestations of cancer of the oral mucosa lesions are leukokeratosis. The inhomogeneous clinical aspect and the larger size of 200mm2 in non-keratinized mucosa as the floor of the mouth and constipation of the tongue, are aspects that imply the possibility of evolution of the lesion. The difficulty of diagnosis of early lesions is in the selection of the site to be biopsied primarily against large and heterogeneous lesions. The objective of this research is to evaluate the use of clinical examination along with diagnostic aids leucoqueratósica of oral lesions (VELscope ®, Toluidine blue and Lugol solution) methods contributes to greater accuracy of diagnosis of dysplasia in these lesions when compared the histopathological. And identify the morphological patterns of these lesions when evaluated by reflectance confocal microscopy. 30 patients older than 18 years, with clinical lesion compatible with oral leukoplakia screened in the outpatient discipline of Clinical Dentistry, Faculty of Dentistry, University of São Paulo and that needed to be biopsied to establish the final diagnosis were selected. Tests toluidine blue, Lugol\'s solution, VELscope ®, reflectance confocal microscopy (RCM) and subsequently, incisional biopsy to obtain the final diagnosis were performed. Patients enrolled had a mean age 60.66 years, 70% (21/30) were female and 30% (9/30) were male. Smoking was reported by 16.7% (5/30) of patients, 60% (3/5) homens.A association of smoking and alcohol use was reported in 10% (3/30) of patients, smoking alone by 6.6% (2/30) and of alcoholism by 3.3% (1/30). The association of habits and alcoholism alone were reported by men only. While the habit of smoking alone was reported by women only. Using the VELscope ® all lesions showed loss of tissue fluorescence, so being all positive for this method. With Lugol\'s solution, 100% of the lesions did not stain, thus being positive for this method. Histopathological examination allowed the diagnosis of 30% (9/30) cases of squamous cell carcinoma, 10% (3/30) of intraepithelial neoplasia, 20% (6/30) of oral lichen planus and 40% (12/30) chronic oral lichen simple. For the use of Lugol VELscope ® and a sensitivity of 100% and a specificity of 50% was observed for the toluidine blue a sensitivity of 63.15%- 66.66% was observed and a specificity of 75.67% - 82.35%. Confocal Microscopy In reflective patterns observed for the four different diagnoses were defined. Auxiliary diagnostic methods chromoscopy and fluorescence mucosa were effective in aiding the delimitation of the extent of injury and Reflectance Confocal Microscopy allowed the visualization of morphological patterns of white lesions noninvasively. Use the Reflectance Confocal Microscopy also allowed the determination of morphological patterns to epidermoid carcinoma, intraepithelial neoplasia, oral lichen planus and lichen simplex chronic oral and proved efficient when compared to histopathology.

碩士<br>國立臺灣大學<br>預防醫學研究所<br>94<br>[Background] Oral squamous cell carcinoma is now the seventh highest type of malignancy in Taiwan. Incidence of oral cancer has continued to increase parallel to the consumption of betel quid since 1980s and with a similar but lesser extent of the consumption of tobacco. Long preclinical detectable phase in oral permalignant lesions (OPL), including oral submucous fibrosis, homogenous leukoplakia, non-homogenous leukoplakia, and erythroplakia, almost preceded the advent of clinical manifest oral cancer. Consequently, with proper screening, these OPLs could be detected early and proper intervention given, hence in hope of reducing incidence of oral cancer. [Study Purpose] (1) To assess whether oral screening with visual inspection with a supplement of Toulidine blue vital dye staining targeting at high risk group (betel quid chewing or smoking) can detect more oral premalignant lesions (2) To quantify the disease natural history for oral cancer and performed a simulation for the cost-effectiveness analysis [Designs and Methods] A community-based randomized controlled trial was designed and conducted in Keelung City in northern Taiwan during 1999. Subjects aged no less than 15 years old with habit of smoking or betel quids chewing were enrolled and randomly assigned into two arms. The experiment group was screened with Toluidine blue dye oral gargling following by visual inspection by experienced oral surgeons. The control group had only placebo dye gargling following by visual inspection by the same blinded oral surgeons. If suspicious oral lesions were identified, referral to tertiary referral medical center for clinical diagnosis and biopsy was arranged. The pathologic report was used as the gold standard for definite diagnosis. Linkage to death registry databank until December 31, 2004 was made to acquire data on occurrence of oral cancer, status of survival and cause of death. Analysis was done on an intention to treat basis. [Results] Of the 8,101 eligible participants recruited in this study, one hundred and twenty-six participants were excluded in the analysis owing to incomplete data collection. A total of 4,080 (51.16%) individuals were included in the experiment group, and 3,895 (48.84%) individuals in the control group. Three hundred and eighty-nine subjects (9.53%) in the experiment group yielded positive screening result while 322 subjects (8.27%) in the control group. After excluding false positive subjects, the positive OPL detection rate was not statistical significant (p=0.64) between the experiment group (4.58%, 187/4,080) and the control group (4.36%, 170/3,895). In subgroup analysis, the detection rate ratio of submucous fibrosis was 1.81 (95%CI 1.05~3.12) between the experiment (41/4,080, 1%) and the control (22/389, 0.56%) group. The relative malignant transformation rate or OPL was 1.29×10-3 in the experiment group and 4.20×10-3 in the control group. The ratio between two groups was 3.25 (95%CI 0.34~31.23) which mean the experiment group tended to detect OPL with higher malignant transformation potential. As for the occurrence oral cancer, it was 11.3×10-5 in the experiment group and 18.45×10-5 in the control group. [Conclusions] The current study is the first one to demonstrate that under the aid of Toluidine blue dye vital staining, mass screening for the high risk subjects could not detect statistically significantly more OPL than screening with visual inspection. The experiment group (41/4,080, 1%) tended to has higher detection rate on oral submucous fibrosis than the control group (22/3,895, 0.56%, p=0.03). In terms of economics, it was a convenient, safe and cost-effective adjunct in mass screening for oral premalignant lesions. Further research with larger cohort or longer follow-up period should be considered to delineate the results more in depth.

碩士<br>國立臺灣大學<br>醫學檢驗暨生物技術學研究所<br>99<br>Like the appearance of methicillin-resistant Staphylococcus aureus (MRSA), the developments of antibiotics-resistant events among a variety of bacteria are due to antibiotics abuses. Drug-resistant strains spring up like mushrooms that indicates antibiotics therapy not enough. To solve these problems, photodynamic inactivation is other anti-microbial strategies. Photodynamic inactivation employs visible light of appropriate wavelength to excite the photosensitizer and then the photosensitizer goes through a transition from ground state to triplet excited state. The triplet excited photosensitizer generates free radicals from electron transfer or reacts with oxygen molecules to produce singlet oxygen. Free radicals and singlet oxygen are toxic to bacteria because of destroying their components such as cell wall, cell membrane and nucleotides. Chitosan is from chitin undergoing deacetylation, and it exhibits antimicrobial effects. In this study, we investigated whether TBO combined with chitosan showed synergistic photodynamic inactivation. The trends of increasing prevalence of MRSA infections worldwide indicate that there are more challenges of MRSA therapy. Biofilms are 100 to 1,000 times less susceptible to antibiotics than planktonic bacteria, so it is difficult to handle biofilm prevention. This study focused on Staphylococcus aureus; one was ATCC 29213 (methicillin-sensitive S. aureus), another was ATCC 33592 (MRSA), and the others were four clinical MRSA strains. The photosensitizer was chosen cationic toluidine blue O (TBO) and then we used red LED light to excite it for evaluating the synergistic photodynamic inactivation efficiency of TBO combined with chitosan against planktonic and biofilm cells. In addition, this study also explored the influence of protease activity under TBO combined with chitosan treatment. The synergistic photodynamic inactivation for S. aureus planktonic cells could be detected under the conditions of low concentration of TBO; however, the effect for biofilm cells was only observed when TBO doses were increased. The synergistic photodynamic inactivation that was supposed to be associated with various genetic regulation and characteristics of bacterial stages affected biofilms less than planktonic cells. According to the results, there was no obvious difference of the synergistic photodynamic inactivation efficiency between methicillin-susceptible or resistant strains including diverse SCCmec types. Thus, the synergistic photodynamic activity by TBO combined with chitosan was shown in all of the tested S. aureus strains. Moreover, chitosan had the ability to inhibit protease activities of S. aureus.

碩士<br>國立臺灣大學<br>醫學檢驗暨生物技術學研究所<br>95<br>The worldwide rise of antibiotic resistance in bacteria has led to the development of new anti-microbial strategies. Photodynamic therapy (PDT) is one of the choices. Photodynamic therapy is based on the combination of photosensitizer, oxygen and harmless visible light to produce cytotoxicity via generation of reactive oxygen species. We studied the photodynamic inactivation (PDI) effects of hematoporphyrin (Hp) and liposome-encapsulated-Hp (Lipo-Hp and polyethylene glycol (PEG) coating Lipo-Hp) on Group A streptococcus (GAS). PDI with 5 μM of Hp or Lipo-Hp followed by light irradiation of 90 J/cm2, resulted in more than 7.4 log killing for GAS. At the same concentration of photosensitizers (0.5μM), and light dose (90 J/cm2), PEG-Lipo-Hp attained 7.2 log killing and Lipo-Hp attained 4.4 log killing for GAS, but Hp only exhibited 0.3 log killing for GAS. The results showed that using lipo-Hp or PEG coating Lipo-Hp as photosensitizers can promote a more efficient photoinactivation of GAS. At the same time, we found out Hp-PDI reduced virulence factor- protease production of GAS. We also studied the toluidine blue O (TBO) and rose bengal (RB) mediated PDI in clinical multidrug resistant bacteria such as vancomycin-resistant Enterococcus (VRE), multidrug-resistant Pseudomonas aeruginosa (MDRPA), and Acinetobacter baumannii. In this study, we show that of TBO-PDT is effective against all strains we used. PDI efficacies were independent of the antibiotic resistance pattern of the given microbial strains. At last, we used rapidly screening method to demonstrate the TBO-PDI activity in 33 multidrug resistant Pseudomonas aeruginosa and 19 susceptible Pseudomonas aeruginosa clinical isolates. We found that minimal lethal drug concentrations were not different between clinical multidrug resistant and susceptible bacteria strains. Our studies have demonstrated that low dose liposome-encapsulated-Hp can enhance the photobactericidal activity of Hp on GAS, and its effect against antibiotic resistant bacteria was independent of their resistance pattern. These promising results suggest that PDI has the potential to be a possible alternative to antibiotic in clinical applications. In the future, we will focus on membrane proteins and nucleic acid damage during Hp-PDT on Group A streptococcus. We hope that we can better understand the mechanisms of photoinactivation for bacteria.

博士<br>國立臺灣大學<br>醫學檢驗暨生物技術學研究所<br>96<br>The VIM-type metallo-β-lactamase (MBL) blaVIM-3 was found in multidrug- resistant (MDR) Pseudomonas aeruginosa clinical isolates from Taiwan. We identified a Tn501-related (class II) transposon, termed Tn6001, which carries a class 1 integron In450-associated multiple drug resistance genes including blaVIM-3. Tn6001 backbone which contains the tnpA, tnpR at the region of left-end and orfB, orfC, orfD at the region of right-end is very similar to that found in Tn1404* or Tn1403. In450 gene cassette contains the following six genes: the blaVIM-3 (resistance to carbapenems), orf2 (putative fosfomycin resistance gene), two copies of aacA4 (resistance to aminogylcoside), aadB (resistance to aminogylcoside), and another copy of aacA4. In450 is related to the In4-like family which contains the 5''-conserved segment (5''-CS), 3''-CS segment and one copy of IS6100. Sequence analysis revealed that In450 is integrated upstream of the tnpR gene in the site II of the res site and lacks the site I. This finding is unlike In4 and In28 with part of res site on each side and with a 5 bp duplication of backbone sequences. I-CeuI digested pulsed-field gel electrophoresis (PFGE) analysis revealed the blaVIM-3 gene is located on the chromosome. Further, hybridization with the blaVIM-3 revealed only one band difference between an imipenem susceptible and an imipenem resistant isolate recovered from the same patient, suggesting that the gene element might have transferred horizontally. Subsequently, we investigated the antibiotic and MBLs surveillance of 316 non-duplicate isolates of carbapenem intermediately-resistant or resistant P. aeruginosa at the National Taiwan University Hospital (NTUH) from 2000 to 2005 and 26 MDR P. aeruginosa isolates collected from different parts of Taiwan from March to August 2003. Overall, 6% (18/316) colistin-only- sensitive (COS) P. aeruginosa and 4% (13/316) pan-drug-resistant (PDR) P. aeruginosa were found in NTUH and 35% (9/26) MDR P. aeruginosa isolates from six hospitals were found colistin intermediately-resistant. 33% (6/18) COS P. aeruginosa and 38% (5/13) PDR P. aeruginosa isolates carried the Tn6001 which contained a blaVIM-3-harboring integron In450 were found in different genotypes at NTUH. These finding suggest that mobile element Tn6001 might have transferred horizontally and cause the resistant genes delivery in Taiwan. MDR Pseudomonas aeruginosa infection is becoming a critical problem worldwide. Currently only limited therapeutic options are available for the treatment of infections caused by MDR P. aeruginosa, therefore the development of new alternative treatments may be needed. In this study, we evaluated the antibacterial photodynamic inactivation effect on MDR P. aeruginosa using toluidine blue O (TBO) as photosensitizer followed by red light irradiation. We demonstrated 4 to 6 logs of killing effect in MDR P. aeruginosa clinical isolates. We also found that TBO caused DNA fragmentation and protein carbonylation in both MDR and susceptible P. aeruginosa clinical isolates. These results suggest that TBO is able to permeate through the membrane and subsequently induces photodamages to DNA and proteins after red light irradiation. Although eleven strains of MDR P. aeruginosa contain an active efflux pump which can pump TBO out, the efficacy of PDT among these resistant strains did not differ from those susceptible strains as shown by minimum lethal drug concentrations (MLC) assay. In summary, antimicrobial photodynamic therapy with TBO showed the same efficacy between efflux pump-positive MDR P. aeruginosa strains and non-MDR strains.

Dissertação de mestrado em Bioinformática<br>Eutrophication is a serious problem of aquatic ecosystems that can threaten biodiversity and human health. It is caused by a high concentration of nutrients, such as phosphorus, leading to excessive growth of aquatic plants and, consequently, to stagnation and decreased levels of oxygen in water systems. To prevent this problem, wastewater from rural, industrial and urban backgrounds are treated using enhanced biological phosphorous removal. This technique uses microorganisms already present in wastewater that incorporate phosphorous into their cells in the form of polyphosphate, removing the nutrient from the water. A very important part of this technique consists of evaluating the polyphosphate levels present in the microorganisms, which is usually done through expensive or time consuming analytical methods. To improve the polyphosphate measurement step, the objective of this thesis was to create a quantitative image analysis program that can correlate image obtained data with analytical data, creating linear regression models useful for polyphosphate quantification. This program used color segmentation between polyphosphate and biomass regions, based on RGB, HSV and LAB color spaces, and was applied on images of samples dyed with different stains (methylene blue and toluidine blue) and with different dilution factors (no dilution and 10x dilution). These images experienced gamma values and contrast adjustments, to evaluate different image settings on color segmentation. Models for methylene blue stained and diluted samples presented the highest regression coefficients between the image data results and analytical data (0.920 and 0.992, respectively), also existing some differences between models of images with different contrast and gamma values. The color space chosen for image analysis seems to be very important with RGB and LAB presenting more satisfying results than HSV. More work is needed before this method can be used, however the results seen on this thesis are promising.<br>A eutrofização é um grave problema de ecossistemas aquáticos que pode ameaçar a biodiversidade e a saúde humana. É causada por uma alta concentração de nutrientes, tal como fósforo, levando a um crescimento excessivo de plantas aquáticas e, consequentemente, à estagnação e diminuição dos níveis de oxigênio nos sistemas de água. Para evitar este problema, as águas residuais das zonas rurais, industriais e urbanas são tratadas com recurso a remoção biológica de fósforo melhorada. Esta técnica utiliza microrganismos já presentes em águas residuais que incorporam o fósforo dentro das suas células na forma de polifosfato, removendo assim o nutriente da água. Uma parte muito importante desta técnica consiste em avaliar os níveis de polifosfato presentes nos microrganismos, o que é geralmente efetuado por métodos analíticos caros e demorados. Para melhorar a etapa de medição de polifosfatos, o objetivo desta tese foi criar um programa de análise quantitativa de imagem que pudesse correlacionar dados obtidos de imagens com dados analíticos, criando modelos de regressão linear úteis para a quantificação de polifosfatos. Este programa utilizou segmentação de cores entre regiões de polifosfato e biomassa, com base nos espaços de cores RGB, HSV e LAB, e foi aplicado em imagens de amostras tingidas com diferentes colorações (azul de metileno e azul de toluidino) e com diferentes fatores de diluição (sem diluição e diluição 10x). Estas imagens foram ajustadas nos seus valores de gamma e contraste, para avaliar diferentes configurações de imagem em segmentação de cor. Os modelos para amostras de azul de metileno e amostras diluídas apresentaram os maiores coeficientes de regressão entre os resultados dos dados de imagem e os dados analíticos (0,920 e 0,992, respetivamente), existindo também algumas diferenças entre os modelos de imagens com diferentes contrastes e valores de gamma. O espaço de cores escolhido para a análise de imagens parece ser muito importante, pois o RGB e o LAB apresentam resultados mais satisfatórios do que o HSV. Mais trabalho é necessário antes que este método possa ser usado, no entanto os resultados vistos nesta tese são promissores.

Introdução: Atualmente o cancro oral é o sexto cancro mais comum em todo o mundo. A sua elevada taxa de morbilidade e consequente mortalidade está relacionada com uma deteção da doença em estádios de malignidade avançados. Esta conjuntura acaba por levar ao aumento de complicações no tratamento, o que o torna mais agressivo consoante o grau de malignidade. Por isso o diagnóstico precoce combinado com o tratamento adequado é a chave fundamental para o controlo da doença. Face a esta realidade os profissionais de saúde têm um papel fulcral na deteção precoce de lesões potencialmente malignas ou malignas, sendo por isso um objetivo prioritário na saúde. Para avaliação deste tipo de lesões o diagnóstico definitivo, histopatologicamente fornecido pela biópsia convencional, continua a ser o método imprescindível e de eleição para os profissionais de saúde, no entanto novas técnicas não-invasivas têm surgido como auxiliares de diagnóstico. Objetivos: Esta monografia teve como objetivo obter um conhecimento detalhado acerca do cancro oral, nomeadamente as suas principais particularidades e especificidades de forma a obter uma deteção precoce e consequentemente um prognóstico favorável. Conjuntamente, foi realizado um estudo que recolheu informações sobre o conhecimento geral dos médicos dentistas e estomatologistas portugueses relativamente ao cancro oral, a utilização de modalidades auxiliares de diagnóstico hoje em dia disponíveis, assim como a colheita de dados face ao protocolo de atuação dos clínicos aquando suspeita de lesões potencialmente malignas ou malignas. Materiais e Métodos: Este trabalho teve como base as palavras-chave delineadas e critérios de inclusão e exclusão específicos, com o objetivo de angariar e debater o máximo de informação acerca desta grande associação entre oncologia e medicina dentária, o cancro oral. De forma a dar resposta aos objetivos enunciados associou-se a esta monografia um estudo quantitativo do tipo descritivo e transversal. A recolha de dados foi realizada através de um inquérito em que participaram 324 profissionais de saúde oral. O inquérito, com a totalidade de vinte e uma perguntas, teve como principal objetivo a recolha de informação sobre o conhecimento geral do médico dentista ou estomatologista relativamente ao cancro oral. Após colheita de dados, estes foram tratados e analisados recorrendo a programas informáticos como o Microsoft Office Excel (2010) e o programa Statistical Package for the Social Sciences (IBM© SPSS© Statistics) vs. 22.0 para Windows. Resultados: A amostra selecionada demonstrou ter os conhecimentos necessários e fundamentais quando abordados sobre este tema, estando conscientemente alerta caso suspeitem de uma lesão potencialmente maligna ou maligna. Cerca de 78% dos inquiridos preferem encaminhar o paciente quando perante uma lesão suspeita. Relativamente aos indivíduos que realizam biópsia, 66.7% prefere a técnica excisional. 96% discute com colegas quando suspeita de uma lesão maligna. A maioria dos profissionais (88.9%) refere que nunca utilizou outros métodos auxiliares para além da biópsia. Dos 11.1% que admitem ter utilizado outro método auxiliar de diagnóstico, 58.8% indica o azul de toluidina como método preferencial. 27.2% dos profissionais não considera a biópsia como elemento fundamental. Discussão e Conclusão: Através da pesquisa realizada verificou-se que é fundamental um conhecimento dos sinais e sintomas e a realização de rastreio em consultas de rotina para detetar precocemente lesões potencialmente malignas ou malignas. Por consenso geral dos inquiridos, constatou-se que a história clínica, o exame clínico e a biópsia são os métodos mais eficazes e comummente utilizados a fim de reduzir a morbilidade e mortalidade associada a esta doença. Ainda assim, 27.2% dos profissionais não considera a biópsia como elemento fundamental o que revela ser preocupante. Contudo corroborou-se que a maioria dos inquiridos, mesmo aptos teoricamente, prefere encaminhar ao invés de realizar biópsia.<br>Introduction: Currently oral cancer is the sixth most common cancer worldwide. Its high rate of morbidity and consequent mortality, is related with the detection of this disease already in advanced stages of malignancy. In this context, it ends up leading to increased complications in treatment that becomes more aggressive according to the degree of malignancy. Therefore early diagnosis along with suitable treatment is the fundamental key to control the disease. Due to this reality, health professionals have a key role in early detection of potential malignant or malignant lesions making it a priority goal in health. To evaluate this kind of lesions, the definitive histopathologic diagnosis provided by conventional biopsy, still remains the crucial method of choice for health professionals, although new non-invasive techniques have emerged as auxiliary diagnosis methods. Objective: This paper aims to obtain a detailed knowledge of oral cancer, including its main particularities and specificities in order to obtain an early detection and consequently a favorable prognosis. It has also have been conducted a study that collected information on the general knowledge of portuguese dentists and stomatologists regarding oral cancer, the use of auxiliary diagnostic tools nowadays available, as well as data collection concerning the clinical performance protocol when suspected potentially malignant or malignant lesions. Materials and Methods: This study was based on the keywords and outlined specific inclusion and exclusion criteria, in order to raise and discuss as much information about this great association between oncology and dentistry, which is oral cancer. In order to meet the stated goals, it was associated with this monography a quantitative study of a descriptive and cross-sectional type. Data collection was conducted through a survey involving 324 dental professionals. The survey, with a total of twenty-one questions, aimed to collect information on the general knowledge of dentists regarding oral cancer. After data collection, these were processed and analyzed using computer programs such as Microsoft Office Excel (2010) and the Statistical Package for Social Sciences (SPSS IBM © © Statistics) vs. 22.0 for Windows. Results: The selected sample has demonstrated the necessary and basic knowledge when approached about the subject, consciously being alert if suspected a potentially malignant or malignant lesion. About 78% of respondents prefer to refer the patient when faced with a suspected lesion. Regarding the individuals that perform biopsy, 66.7% prefer the excisional technique. 96% approaches other colleagues when suspects of a malignant lesion. Most professionals (88.9%) reported ever used other auxiliary methods in addition to biopsy. Of the 11.1% who admit to having used an auxiliary diagnostic tool, 58.8% indicates the toluidine blue as the preferred method. 27.2% of professionals do not consider the biopsy as a fundamental element. Discussion and Conclusion: Through the research made, it was found that it is essential to have knowledge of signs and symptoms and provide screening in routine consults aiming for an early detection of potentially malignant or malignant lesions. By general consensus of those surveyed, it was found that the clinical history, clinical examination and biopsy are the most effective methods and commonly used to reduce morbidity and mortality associated with this disease. Still, 27.2% of the respondents do not consider the biopsy as a fundamental element, which is concerning. However it was corroborated that the majority of respondents although theoretically able, prefer to refer instead of performing biopsy.