Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / INTRODUCCIÓN: En el biofilm dental se encuentran diversas bacterias, de las cuales Streptococcus mutans (S. mutans) es considerada como una de las de mayor potencial cariogénico. Existen diversos métodos para el control del biofilm, entre los que se encuentran los métodos químicos. Estos métodos se basan en el uso de sustancias antisépticas y/o antibióticas que son utilizados como colutorios orales y que permiten disminuir o retardar la formación de la placa bacteriana. En la actualidad no existe un agente químico antimicrobiano ideal. Es por esto que constantemente se están realizando cambios a los agentes terapéuticos actualmente conocidos, ya sea mejorando sus propiedades antimicrobianas, sus propiedades de adhesión (sustantividad) o disminuyendo sus efectos adversos. Es aquí donde la Nanotecnología y el desarrollo de nanomateriales han adquirido especial importancia debido a la posibilidad de sintetizar materiales con propiedades antimicrobianas. Es sabido que nanopartículas de quitosano (QuitNP) y nanopartículas de cobre (CuNP) presentan propiedades antimicrobianas frente a diversas bacterias, tanto Gram negativas como Gram positivas. Sin embargo no se ha probado la efectividad de las CuNP sobre S. mutans. Por otra parte estudios demuestran que nanopartículas metálicas, como el cobre, soportadas en una matriz de quitosano, presentarían mejoras en sus propiedades de adhesión. El objetivo de este trabajo de tesis es la síntesis de nanopatículas de cobre y quitosano, y evaluar sus propiedades antimicrobianas frente a S. mutans. MATERIAL Y METODOS: Se sintetizaron CuNP c/a utilizando almidón como agente reductor y estabilizante, CuNP s/a sin utilizar almidón, QuitNP y una nanopartícula hibrida (CuNP/QuitNP). Para esto se usaron reactivos biocompatibles, que permitieron la reducción a nanopartículas metálicas, y la gelificación iónica de nanopartículas poliméricas. Las muestras fueron caracterizadas mediante espectrofotometría de absorción molecular, microscopia electrónica de barrido (SEM), difracción de rayos X (DRX), espectroscopia infrarroja de reflectancia total atenuada (FTIR-ART) y análisis termogravimétricos (TGA). Se realizaron ensayos de actividad bactericida, para lo cual se determinó la concentración inhibitoria mínima (CIM) utilizando el método de incubación de medio de cultivo líquido, y la concentración bactericida mínima (CBM) realizando el recuento de unidades formadoras de colonias (UFC) en placas de agar BHI, para las nanopartículas sintetizadas. Además se realizaron pruebas de actividad bactericida sobre un biofilm formado en superficies dentarias. Para esto se utilizaron fragmentos de terceros molares, con un biofilm formado, los que fueron tratados con distintas soluciones antimicrobianas y suspensiones de nanopartículas. RESULTADOS: Utilizando el concepto de “química verde”, se sintetizaron y caracterizaron las partículas de carácter nanométrico; CuNP c/a, QuitNP y CuNP/QuitNP. Por otro lado, las partículas CuNP s/a presentaron dimensiones micrométricas. En los ensayos de actividad bactericida, las CuNP c/a fueron las que presentaron la menor CIM (20 µg/ml) y CBM (30 µg/ml) en comparación a las otras suspensiones. En los ensayos sobre un biofilm formado sobre una superficie dentaria, aquellas nanopartículas que tenían quitosano como parte de su composición fueron las que presentaron mejores resultados, siendo la nanopartícula hibrida (CuNP/QuitNP), que al probarla frente a S. mutans, se obtuvo el menor recuento de UFC. CONCLUSIÓN: El método estudiado en este trabajo, utilizando reactivos biocompatibles permitió la formación de nanopartículas de cobre y quitosano de manera satisfactoria y con características compatibles para futuros estudios en control de biofilm. La incorporación de CuNP c/a durante el proceso de formación de las QuitNP, permitió la síntesis de una nanopartícula hibrida cobre-quitosano (CuNP/QuitNP). Tanto las nanopartículas de cobre, de quitosano y sus combinaciones presentaron actividad antimicrobiana frente al patógeno Streptococcus mutans. Por otra parte, todas las suspensiones de nanopartículas presentaron un efecto antibacteriano al tratar un biofilm de S. mutans previamente formado sobre una superficie dental.

Autoimmune encephalitic (AE) syndromes represent a unique manifestation of autoimmunity: the immune system recognizes the brain as foreign, and interferes with neuronal function. AE syndromes are characterized by hallucinations, paranoia, anxiety, seizures, and autonomic dysregulation, and progress over a matter of weeks as autoantibodies targeting the brain bind more densely to their CNS targets. Development of AE has been linked to peripheral tumors and infection, both of which provide structural mimetics to CNS antigens to incite an immune response in the periphery. How these brain-specific antibodies reach the CNS remains unclear. In rare cases, Group A Streptococcus (GAS, S. pyogenes) infections can cause CNS autoimmunity targeting the basal ganglia, termed post-infectious basal ganglia encephalitis (BGE), manifesting as motor (Sydenham’s chorea) and psychiatric (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus, PANDAS) abnormalities. In a mouse model of post-infectious BGE, we have shown that repeated intranasal GAS infections generate robust T cell infiltration into the CNS, concomitant with blood-brain barrier breakdown, microglial activation, and olfactory synapse degradation. However, the different T cell subtypes that enter the brain, how they enter the CNS, and their relative contributions to neural dysfunction and neuroinflammation remain unclear. Th17 lymphocytes are heavily implicated in many autoimmune diseases, but whether the inflammatory post-infectious BGE reaction induces functional deficits in odor processing and requires Th17 lymphocytes was unknown. Here we demonstrate that mice lacking Th17 lymphocytes display both reduced BBB impairment and antibody infiltration. Furthermore, multiple GAS infections induce deficits in odor processing, which are partially ameliorated in Th17 cell-deficient mice. Notably, neuroinflammation and some excitatory synaptic loss persist, due to the presence of Th1 lymphocytes. Th17 lymphocytes are therefore critical for both selective CNS entry of autoantibodies and neural circuit impairment during post-infectious BGE. By examining the regulation of chemokine ligand expression in GAS-inoculated mice, we determined that CCL20/CCR6 and CCL2/CCR2 chemokine axes are implicated in T cell homing to the brain. In chemokine receptor mutant CCR6+/- CCR2-/- mice, T cell infiltration is reduced by 86.2%, and microglial activation is blunted. These findings suggest that CCL2/CCR2 and CCL20/CCR6 signaling may play a role in T cell homing to the brain and neuroinflammation. Finally, we first assessed behavioral and immune responses in two different GAS exposure models. It was clear that while behavioral abnormalities can be recapitulated in mice given subcutaneous GAS immunizations, this elicited relatively weak cellular and humoral immune responses. By contrast, mice given intranasal GAS inoculations showed minimal behavioral abnormalities, but elicited robust humoral and cellular immune responses. Taken together, these data demonstrate the pivotal role of Th17 lymphocytes in brain pathology and olfactory processing deficits after recurrent GAS infections in our mouse model. Our intranasal inoculation model supports the conclusion that post-infectious BGE is autoimmune in nature, despite the absence of behavioral symptoms in this model. Using multiple mouse models of post-infectious BGE may allow us to study distinct facets of disease pathogenesis. Finally, this work underscores the ability of T cells to incite neuroinflammation, provides a useful clinical diagnostic test in olfactory functional assessments, and lends support to T cell immunotherapy strategies in patients with post-infectious BGEs.

DSc (Med), Faculty of Health Sciences, University of the Witwatersrand, 2009.

The serum of an immunized animal will contain antibodies referred to as agglutinins, precipitins, opsonins, bacteriolysins, or complement-fixing antibodies (Zinsser 1952). The presence of such antibodies may be demonstrated in the laboratory, the type of reaction depending on the circumstance and the laboratory manipulation employed. Regardless of the specific serolological method utilized, the manifestation of the antigen-antibody reaction is the visible observation that such a combination has occurred.

Um novo monômero, brometo de metacriloiloxidodecilpiridínio (MDPB), com efeito antibacteriano e capacidade de co-polimerizar com outros monômeros, foi apresentado por Imazato, Torii e Tsuchitani (1993). Este estudo avaliou a adesão bacteriana, em 16, 40 e 64 horas, à resina composta contendo ou não o monômero antibacteriano MDPB. A adesão foi testada para Streptococcus sanguinis e Streptococcus sobrinus. Após as amostras terem sido submetidas à incubação, o biofilme foi coletado e a contagem de UFCs foi realizada. Os dados foram comparados pelo método ANOVA complementado por teste de Tukey. Os resultados demonstraram que, para o S. sanguinis, a adesão sobre o MDPB foi significativamente maior (p<0.05) quando comparado ao controle em 16 horas, mas diminuiu significativamente em 40 horas, não apresentando diferenças quando comparado ao controle neste tempo (p<0.01). Para o S. sobrinus, o controle apresentou aumento significativo da adesão bacteriana em 64 horas quando comparado com 16 horas (p<0.01), sendo significativamente maior que para o MDPB em 64 horas (p<0.05). Assim, o estudo mostrou que o MDPB é capaz de inibir a adesão de S. sobrinus sem interferir na adesão do S. sanguinis. Portanto, nas condições deste estudo os resultados sugerem que a incorporação do MDPB a resinas compostas pode ser de importância na prevenção de cáries secundárias favorecendo a adesão de bactérias comensais em detrimento de bactérias com potencial cariogênico. / A new antibacterial monomer, Methacryloyloxydodecylpyridinium bromide (MDPB), with antibacterial property and ability to co-polymerize with other monomers, was introduced by Imazato, Torii and Tsuchitani (1993). This study aimed to analyze the effect of MDPB on bacterial adherence to resin composites containing or not MDPB. Streptococcus sanguinis and Streptococcus sobrinus were used. The biofilms were collected from the samples and the colony forming units (CFUs) were counted after 16, 40 e 64 h of incubation. The data were compared by ANOVA complemented by the Tukey’s test. The results showed that the adhesion of S. sanguinis to MDPB-containing resin composite was significantly higher (p<0.05) than to control samples at 16 h, but significantly diminished at 40 h, reaching values similar to those of control samples (p<0.01). The adherence of S. sobrinus to control samples significantly increased throughout the experimental time (p<0.01) and was considerably higher than to MDPB at 64 h (p<0.05). Thus, the study showed that MDPB is capable of inhibit adhesion of S. sobrinus with no interference on the adhesion of S. sanguinis. Thus, at the conditions of this study we suggest that MDPB incorporated to resin composites could be of importance to prevent secondary caries favoring adhesion of commensal bacteria and impairing adhesion of cariogenic bacteria.

Streptococcus pneumoniae is a highly diverse and adaptable opportunistic pathogen that can infect and colonise different niches within the human host to cause a wide range of invasive disease (sepsis and meningitis) and noninvasive disease (pneumonia, otitis media and sinusitis). The molecular mechanisms that contribute to the different patterns of pneumococcal infection remain largely unknown. This thesis aims to determine the physiological and proteomic responses that allow the pneumococcus to survive and adapt to invasive and non-invasive sites. The comparative proteomic analyses of clinical S. pneumoniae isolates recovered from blood cultures (classified as invasive site isolates) and mucosal surfaces such as sputum, skin and ear swabs (classified as non-invasive site isolates) was initiated. The pneumococci were grown in vitro under standard conditions and the total cellular bacterial proteins extracted and analysed using both gel based and non-gel based proteomic approaches. Analysis of the pneumococcal isolates by two-dimensional polyacrylamide gel electrophoresis (2DGE) revealed that a high degree of heterogeneity existed between the pneumococcal isolates particularly among isolates in the invasive site isolates. Differential patterns of protein synthesis were observed that discriminated the pneumococcal isolates according to their sites of isolation. These were proposed to be associated with the bacterial adaptation to invasive and non-invasive sites of infection. Mass spectrometry was used to identify selected significant (ANOVA, p < 0.05) protein spots, which were further categorised into functional groups by Gene Ontology analysis. An extension of the 2DGE data using an integrated approach comprising bioinformatics, surfome analysis and a shotgun proteomic workflow provided a comprehensive qualitative and quantitative analyses of the pneumococcal intracellular and cell-surface proteomes. Proteins potentially involved in pneumococcal niche-specific adaptation and surface proteins with potential for further investigation and inclusion in the pipeline of vaccine candidates were identified. Quantitative regulation of proteins involved in energy metabolism, genetic competence, stress response, surface adhesion and virulence were considered important for pneumococcal adaptation to invasive and non-invasive sites. The anatomical sites colonised by the pneumococcus vary in their V availability for iron. The 2DGE method was also used on selected pneumococcal isolates from the two sites of infection to define the proteome variability linked to the effect of iron starvation that may contribute to the different disease outcomes associated with pneumococcal infections. The iron restricted condition was generated by cation depletion of the growth medium using Chelex-100. Quantitative differences in protein abundance were demonstrated that correlated with pneumococcal adaptation to iron restriction. The identification of selected significant spots by liquid chromatography-mass spectrometry and systems biology analysis of the identified proteins contributed to the elucidation of the molecular mechanisms underlying pneumococcal survival under iron limitation. The expression/repression of proteins functionally associated with metal ion binding, oxidative stress response, translation and virulence mainly constituted the pneumococcal adaptive responses to growth under conditions of limited iron availability. The data presented in this thesis extended our understanding of the molecular events underlying pneumococcal physiological adaptation and provide the basis of future work in this area.

Cheuk Shing Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 144-161). / Abstracts in English and Chinese. / ACKNOWLEDGMENTS --- p.I / 內容摘要 --- p.II / ABSTRACT --- p.IV / CONTENTS --- p.XI / LIST OF TABLES --- p.XI / LIST OF FIGURES --- p.XI / ABBREVIATIONS --- p.XII / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Taxonomy of Streptococcus agalactiae --- p.1 / Chapter 1.2 --- Characteristics of Streptococcus agalactiae --- p.1 / Chapter 1.3 --- Epidemiology of GBS --- p.3 / Chapter 1.3.1 --- Risk groups --- p.3 / Chapter 1.3.1.1 --- Neonates --- p.3 / Chapter 1.3.1.2 --- Pregnant women --- p.5 / Chapter 1.3.1.3 --- Non-pregnant adult --- p.6 / Chapter 1.3.2 --- World wide distribution --- p.7 / Chapter 1.3.2.1 --- Serotypes --- p.7 / Chapter 1.3.2.2 --- Antibiotic susceptibility --- p.8 / Chapter 1.3.3 --- GBS diseases in Hong Kong --- p.10 / Chapter 1.4 --- Putative virulence factors and pathogenesis --- p.10 / Chapter 1.4.1 --- Capsular polysaccharide --- p.10 / Chapter 1.4.2 --- C5a peptidase --- p.11 / Chapter 1.4.3 --- β-haemolysin/cytolysin --- p.12 / Chapter 1.4.4 --- C protein and C a-like protein --- p.12 / Chapter 1.4.4.1 --- C protein --- p.12 / Chapter 1.4.4.1.1 --- C α protein --- p.13 / Chapter 1.4.4.1.2 --- Cβ protein --- p.14 / Chapter 1.4.4.2 --- C α-like protein --- p.15 / Chapter 1.4.5 --- Hyaluronate lyase --- p.16 / Chapter 1.4.6 --- CAMP factor --- p.17 / Chapter 1.4.7 --- Others --- p.17 / Chapter 1.5 --- Antibiotic resistance and resistance genes --- p.18 / Chapter 1.5.1 --- Macrolides --- p.18 / Chapter 1.5.2 --- Tetracyclines --- p.18 / Chapter 1.5.3 --- Aminoglycosides --- p.19 / Chapter 1.5.4 --- Fluoroquniolones --- p.20 / Chapter 1.5.5 --- Others --- p.20 / Chapter 1.6 --- Mobile genetic elements --- p.21 / Chapter 1.7 --- Typing methods --- p.22 / Chapter 1.7.1 --- Phenotypic methods --- p.23 / Chapter 1.7.1.1 --- Serotyping --- p.23 / Chapter 1.7.1.2 --- Multilocus enzyme electrophoresis (MLEE) --- p.23 / Chapter 1.7.2 --- Genotypic methods --- p.24 / Chapter 1.7.2.1 --- Restriction endonuclease analysis (REA) / restriction fragment-length polymorphism (RFLP) --- p.24 / Chapter 1.7.2.2 --- Pulsed-field gel electrophoresis (PFGE) --- p.25 / Chapter 1.7.2.3 --- Random amplified polymorphic DNA (RAPD) --- p.26 / Chapter 1.7.2.4 --- Sequencing --- p.26 / Chapter 1.8 --- Prevention --- p.29 / Chapter 1.8.1 --- Intrapartum antibiotic prophylaxis (IAP) --- p.29 / Chapter 1.8.2 --- GBS Vaccine --- p.33 / Chapter 1.9 --- Objectives --- p.34 / Chapter CHAPTER 2 --- METHODS AND MATERIALS --- p.35 / Chapter 2.1 --- Bacterial isolates --- p.35 / Chapter 2.2 --- Antibiotic susceptibility test --- p.37 / Chapter 2.2.1 --- Antibiotic preparation --- p.37 / Chapter 2.2.2 --- Microbroth dilution method --- p.39 / Chapter 2.2.2.1 --- Microtitre plate preparation --- p.39 / Chapter 2.2.2.2 --- Suspension preparation and inoculation --- p.39 / Chapter 2.2.2.3 --- End points determination --- p.40 / Chapter 2.2.3 --- Inducible lincomycin resistance determination --- p.40 / Chapter 2.3 --- Serotyping --- p.41 / Chapter 2.3.1 --- Preparation of antigens --- p.41 / Chapter 2.3.2 --- Typing of isolates --- p.42 / Chapter 2.4 --- Pulsed-field Gel Electrophoresis (PFGE) --- p.42 / Chapter 2.4.1 --- Preparation of DNA plug for PFGE --- p.43 / Chapter 2.4.2 --- Restriction enzyme digestion of GBS DNA --- p.43 / Chapter 2.4.3 --- Running of PFGE gel --- p.44 / Chapter 2.5 --- Molecular characterization --- p.44 / Chapter 2.5.1 --- Target genes --- p.44 / Chapter 2.5.2 --- DNA preparation --- p.51 / Chapter 2.5.3 --- Master mix preparation --- p.51 / Chapter 2.5.4 --- Polymerase chain reaction --- p.51 / Chapter 2.5.5 --- PCR product analysis by agarose gel electrophoresis --- p.52 / Chapter 2.5.6 --- DNA sequencing --- p.52 / Chapter 2.6 --- Data analysis --- p.53 / Chapter 2.6.1 --- PFGE and molecular characters analysis --- p.53 / Chapter 2.6.2 --- Sequences analysis --- p.53 / Chapter 2.7 --- Molecular identification by real-time PCR --- p.54 / Chapter 2.7.1 --- Bacterial strains --- p.54 / Chapter 2.7.2 --- DNA isolation for specimens --- p.56 / Chapter 2.7.3 --- Design of TaqMan primers and probes --- p.56 / Chapter 2.7.4 --- Cloning of target sequences --- p.59 / Chapter 2.7.5 --- Master mix of real-time PCR --- p.59 / Chapter 2.7.6 --- Specificity and detection limit --- p.60 / Chapter CHAPTER 3 --- RESULTS --- p.62 / Chapter 3.1 --- Serotype distribution of GBS --- p.62 / Chapter 3.1.1 --- Serotyping using antisera --- p.62 / Chapter 3.1.2 --- Serotyping by molecular method --- p.64 / Chapter 3.1.3 --- Molecular subtype of GBS serotype III --- p.66 / Chapter 3.1.4 --- Correlation of serotypes with diseases --- p.69 / Chapter 3.2 --- Antimicrobial susceptibility --- p.71 / Chapter 3.2.1 --- Phenotypic method --- p.71 / Chapter 3.2.2 --- Detection and distribution of resistance genes --- p.76 / Chapter 3.2.2.1 --- Tetracycline resistance --- p.76 / Chapter 3.2.2.2 --- Macrolide and lincosamide resistance --- p.77 / Chapter 3.2.2.3 --- Aminoglycoside resistance --- p.78 / Chapter 3.3 --- Molecular typing --- p.83 / Chapter 3.3.1 --- Pulsed-field gel electrophoresis (PFGE) --- p.83 / Chapter 3.3.2 --- Distribution of GBS surface protein genes profiles --- p.89 / Chapter 3.3.3 --- Distribution of mobile genetic elements --- p.92 / Chapter 3.4 --- "Analysis based on PFGE, surface protein genes, mobile genetic elements and antibiotic resistance genes" --- p.95 / Chapter 3.4.1 --- Intra-molecular serotype --- p.95 / Chapter 3.4.1.1 --- Molecular serotype Ia --- p.95 / Chapter 3.4.1.2 --- Molecular serotype Ib --- p.99 / Chapter 3.4.1.3 --- Molecular serotype II --- p.101 / Chapter 3.4.1.4 --- Molecular serotype III --- p.103 / Chapter 3.4.1.5 --- Molecular serotype V --- p.107 / Chapter 3.4.1.6 --- Molecular serotype VI --- p.110 / Chapter 3.4.1.7 --- "Molecular serotype IV, VII and VIII" --- p.110 / Chapter 3.4.1.8 --- Non-typeable isolate (NT) --- p.111 / Chapter 3.4.2 --- Analysis of Maternal and neonatal strains --- p.115 / Chapter 3.4.3 --- Comparison of GBS strains from Hong Kong to Australia and Korea --- p.118 / Chapter 3.5 --- Molecular identification of GBS by real-time PCR --- p.120 / Chapter 3.5.1 --- Specificity --- p.120 / Chapter 3.5.2 --- Detection limits --- p.122 / Chapter CHAPTER 4 --- DISCUSSION --- p.125 / Chapter 4.1 --- Laboratory methods for typing and characterization of GBS --- p.125 / Chapter 4.1.1 --- Serotyping by agglutination and molecular method --- p.125 / Chapter 4.1.2 --- Antibiotic susceptibility testing and resistance genes --- p.129 / Chapter 4.1.3 --- PFGE --- p.130 / Chapter 4.1.4 --- Surface protein genes --- p.131 / Chapter 4.1.5 --- Mobile genetic elements --- p.132 / Chapter 4.1.6 --- Real-time PCR --- p.133 / Chapter 4.2 --- Characterization of GBS in Hong Kong --- p.135 / Chapter 4.2.1 --- GBS in Hong Kong --- p.135 / Chapter 4.2.2 --- GBS from Australia and Korea --- p.141 / Chapter 4.3 --- Future research --- p.142 / Chapter 4.4 --- Conclusions --- p.143 / REFERENCES --- p.144 / APPENDIX I: MATERIALS AND REAGENTS --- p.162 / APPENDIX II: DENDROGRAMS --- p.168

Orientador: Osmir Batista de Oliveira Junior / Banca:Rosane de Fatima Zanirato Lizarelli / Banca: Sergio Luiz de Souza Salvador / Banca: Rita de Cassia Loiola Cordeiro / Banca: Marcelo Ferrarezi de Andrade / Resumo: O uso indiscriminado dos antibióticos e seu mecanismo de ação levaram ao desenvolvimento de cepas bacterianas altamente resistentes e de maior virulência. Estas cepas causam doenças muito mais agressivas e de difícil tratamento, constituindo-se um dos principais desafios enfrentados por profissionais da área da saúde. Como a terapia fotodinâmica antimicrobiana (TFDA) baseia seu mecanismo de ação em reações oxidativas não especificas, esta passou a ser uma alternativa interessante para o tratamento de todas as doenças de origem microbiana, uma vez que, nem bactérias, nem vírus ou fungos tem capacidade de desenvolver resistência a TFDA. Considerando que a carie dental é uma doença de origem bacteriana especifica e que a eficácia da TFDA depende do tipo e dose de energia luminosa utilizada, do fotossensibilizador e da taxa de oxigênio nos tecidos a serem tratados, decidimos investigar a viabilidade da TFDA para prevenção e tratamento da cárie dental. Para tal, foram realizados 3 estudos: 1) Revisão critica da literatura sobre fotossensibilizadores utilizados para controle antimicrobianos de Streptococcus mutans. 2) Avaliação do efeito antibacteriano de três fotossensibilizadores (curcumina, hipericina e hematoporfirina) sobre Streptococcus mutans em suspensões planctônicas e 3) Avaliação do efeito antibacteriano de três fotossensibilizadores (curcumina, hipericina e photogem®) Esteban Florez FL. Terapia Fotodinâmica em microrganismos cariogênicos - Estudo in vitro. [Tese de doutorado]. Araraquara: sobre biofilme maduro formado a partir de cepa de Streptococcus mutans. Dos fotossensibilizadores utilizados a hipericina foi a que apresentou maior efeito antimicrobiano sobre suspensão planctônica. Foi comprovado que a eficácia da TFDA esta diretamente relacionada com o tipo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The indiscriminate use of antibiotics and its mechanisms of action led to the development of highly resistant bacterial strains and more virulent ones. This strains can cause diseases much more aggressive and difficult to treat, and in that way they consist in one of the major challenges to the health care professionals. As the antimicrobial photodynamic therapy (APDT) it is based on non-specific oxidative reaction, this is now considered as an interesting alternative to treat all diseases from bacterial origins, once that, neither bacterias, neither viruses nor fungi can develop acquired resistance from the therapy. Taking into consideration that dental caries is a specific bacterial disease and that the APDT's efficiency is directed related to factors as, wavelength, energy dose, photosensitizer used, and with the oxygen present in the target tissue, we have decided to investigate the APDT to prevent and treat dental decay. To accomplish that, it was realized three studies: 1) Critical Literature revision about photosensitizers used to control S.mutans. 2) Evaluation of the antimicrobial effect of three different photosensitizers (Curcumin, Hypericin and Hematoporfirin) over Streptococcus mutans in planktonic cultures and 3) Evaluation of the antimicrobial effect of three photosensitizers (Curcumin, Hypericin and Hematoporfirin) over mature biofilms obtained from Streptococcus mutans strains. From the photosensitizers used Hypericin was the one that showed the most antibacterial observed effect on the planktonic cultures. It was demonstrated by our data that the APDT efficacy is directly related to the time of irradiation of the samples, with the kind of photosensitizer used, its concentration and with the energy dose delivered. None of the proposed protocols were able to show any significant effect over the microorganisms when in biofilm... (Complete abstract click electronic access below) / Doutor

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista / Introducción: La caries es una de las enfermedades más prevalentes a nivel mundial. Se caracteriza por la pérdida de minerales a causa de subproductos ácidos liberados por bacterias presentes en la biopelícula adherida a las superficies de los dientes. Esta produce lesiones que pueden culminar en la pérdida de la estructura dentaria, junto con todas las repercusiones que esto genera en la calidad de vida y salud de las personas. Los agentes remineralizantes a base de flúor son productos utilizados para revertir la pérdida de minerales en las etapas iniciales de esta enfermedad. La finalidad de esta investigación radicó en evaluar la capacidad remineralizante de BlueRemin®, un nuevo agente remineralizante a base de flúor y grafeno, con el objetivo de iniciar una línea de investigación que permita evaluar sus propiedades y futuras aplicaciones clínicas. Objetivos: Establecer diferencias en la dureza superficial en especímenes de esmalte desmineralizados mediante un modelo de caries microbiológico y luego de ser sometidos a un protocolo de remineralización con BlueRemin®. Metodología: Se utilizó el modelo de caries descrito por Ccahuana y Cury (2010), que consiste en la simulación de un ambiente cariogénico mediante la exposición de bloques de esmalte a sacarosa, previa formación de una biopelícula monoespecie de Streptococcus mutans, lo cual permitió obtener una zona de desmineralización compatible con una lesión de caries no cavitada que luego fue remineralizada con el agente remineralizador experimental BlueRemin®. Los bloques de esmalte fueron evaluados por microdureza de Vickers antes de la exposición a sacarosa, después de la desmineralización y una vez aplicado el agente remineralizador. Resultados: Las microdurezas de los especímenes de esmalte expuestos al modelo de caries experimentaron un aumento estadísticamente significativo (p < 0.05) luego de ser tratados con el agente remineralizador BlueRemin®. Conclusiones: Debido a que la aplicación de BlueRemin® generó un aumento de la dureza superficial estadísticamente significativo (p < 0.05) de todas las muestras de esmalte, se sugiere que posee capacidad remineralizante. / Adscrito a Proyecto Open Beaucheff "Remineralizador cariostático basado en flúor y grafeno"

Group B streptococcus (GBS, S. agalactiae) and human ureaplasmas (U. parvumand U. urealyticum) are two clinically and phylogenetically related, potential perinatal pathogens. Their relationships between genotypes and pathogenesis of GBS and ureaplasma infection were still not well understood, one of the reason is that both of them are still short of a very practical genotyping system. In the study, to solve the above problem we developed genotyping systems for the organisms (the second section). For human ureaplasmas, based on four genes/gene clusters (rRNAgene clusters, the elongation factor Tu genes, urease gene complexes and multiplebanded antigen genes), we designed many primer pairs suitable for developing species identification assays for the two newly established human ureaplasma species (U. parvum and U. urealyticum). Further, based on the heterogeneity of ureaplasma multiple banded antigen gene (which contains species- and serovar-specific regions), we developed genotyping methods for each ureaplasma species.For GBS, based on three sets of molecular markers (capsular polysaccharidesynthesis gene clusters, surface protein antigen genes and mobile genetic elements),we developed a genotyping system. The primary evaluation of the genotyping systems showed that the genotyping systems were practical alternative assays for the conventional serotyping and they will be useful to further explore the relationships between genotypes and pathogenesis of GBS and ureaplasma infection. In the study, we introduced novel data and tools into GBS and ureaplasma studies especially from genomic- and bioinformatics-based molecular microbiology(the third section). For two newly established human ureaplasma species, based on the U. parvum serovar-3 genome, and using the above four important genes/geneclusters, we exposed some interesting problems in the understanding of newureaplasma taxonomy especially in the post genomic era. For GBS, we studied the two published full genomes and exposed some new problems or possible future new research fields. In particular we found the two finished and one ongoing GBS genomes were all non-typical and suggest that future genomic project had better have genetic population structure viewpoint. Finally, we suggested that integrated studies of the two potential or conditional perinatal pathogens, from the viewpoint of evolution, would provide a new understanding angle of the pathogenesis of the two organisms. Studies suggested that during coevolution, human ureaplasmas(especially U. parvum) became friendlier than their ancestors to their human host (by losing most of its virulence genes); however, GBS tried to increase its invasive abilities (by getting more virulence genes) to fight against the human host attack.