Submitted by Cássio da Cruz Nogueira (cassionogueirakk@gmail.com) on 2017-05-05T11:38:15Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PeriodontiteOvinosEstado.PDF: 11749160 bytes, checksum: 07582d26b606607e8e1786f83ce34921 (MD5) / Approved for entry into archive by Edisangela Bastos (edisangela@ufpa.br) on 2017-05-09T20:27:18Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PeriodontiteOvinosEstado.PDF: 11749160 bytes, checksum: 07582d26b606607e8e1786f83ce34921 (MD5) / Made available in DSpace on 2017-05-09T20:27:18Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_PeriodontiteOvinosEstado.PDF: 11749160 bytes, checksum: 07582d26b606607e8e1786f83ce34921 (MD5) Previous issue date: 2015-01-29 / O presente trabalho relata os aspectos epidemiológicos, clinico-patológicos e bacteriológicos do primeiro registro de periodontite em ovinos no Brasil, ocorrido em uma propriedade rural no município de Benevides, Para. O surto ocorreu aproximadamente um mês após o pastejo na área de Panicum maximum cv. Massai, a qual sofreu praticas agrícolas, quando foi observado aumento de volume na mandíbula em alguns animais, em sua maioria com idade acima de 36 meses. Foi realizado o exame clinico extra-oral dos 545 ovinos e verificou-se aumento de volume da mandíbula em 3,7%. Os animais acometidos apresentavam baixo escore corporal, pelos arrepiados e sem brilho, alguns com afrouxamento e perda dos dentes pré-molares e molares inferiores e superiores, formação de abscesso e fístula no local acometido, demonstração de dor a palpação e dificuldade de mastigação. Das 39 cabeças de animais jovens analisadas no exame macroscópico post-mortem, 51,3% apresentavam lesões periodontais e das 38 analisadas após a maceração, 73,7% também apresentavam lesões. Das cabeças com lesões no exame post-mortem, em 45% as lesões encontravam-se na maxila, 15% na mandíbula e 40% nas duas estruturas (maxila e mandíbula). Nas cabeças com lesões após a maceração, 50% encontravam-se na maxila e 50% na maxila e mandíbula. Das 17 cabeças de animais adultos analisadas no exame post-mortem e após a maceração, todas apresentavam lesões periodontais. No exame post-mortem, 11,8% apresentavam lesões na mandíbula e 88,2% nas duas estruturas; após a maceração, 5,9% na maxila, 11,8% na mandíbula e 82,3% nas duas estruturas. Os achados histopatológicos revelaram processo inflamatório piogranulomatoso. Para caracterização da microbiota bacteriana subgengival de 14 ovinos com periodontite foi realizada a Reação em Cadeia da Polimerase (PCR) para pesquisa de micro-organismos pertencentes ao complexo vermelho de Socransky (Porphyromonas gingivalis, Tannerella forsythia e Treponema denticola) e outros possíveis periodontopatogenos Gram-negativos e Gram-positivos. Em 50% das amostras foi possível identificar Porphyromonas gingivalis, em 92,8% Tannerella forsythia e em 78,5% Treponema denticola. Os três periodontopatogenos pertencentes ao complexo vermelho ocorreram concomitantemente em 42,8% das amostras. Foram identificados ainda em pelo menos um animal examinado Campylobacter rectus, Eikenella corrodens, Enterococcus faecium, Fusobacterium nucleatum, Prevotella intermedia, Prevotella loeschii e Prevotella nigrescens. Não foram detectados nas 14 amostras Aggregatibacter actinomycetemcomitans, Dialister pneumosintes, Enterococcus faecalis e Porphyromonas gulae. Os resultados permitem concluir que a periodontite ovina ocorrida no município de Benevides – PA possui etiologia infecciosa e acometeu um significativo numero de animais, diversos com abaulamento da mandíbula; teve alta incidência nos jovens e envolveu a totalidade dos animais adultos examinados post-mortem e após a maceração. / This paper describes the epidemiological, clinical, pathological and bacteriological the first periodontitis record in sheep in Brazil occurred in a rural property in the municipality of Benevides, Para. The outbreak occurred about a month after grazing in the area of Panicum maximum cv. Massai, which suffered agricultural practices, there was swelling in the jaw in a few animals, mostly those over the age of 36 months. The extraoral examination was performed in 545 sheep and there was swelling of the jaw in 3.7% of them. Clinical signs were low body score, rough hair coat and dull, some relaxation and loss of premolars and upper and lower molar teeth, abscess and fistula formation in the affected site, demonstration of pain on palpation and difficulty chewing. Between the 39 heads of young animals analyzed in the post-mortem examination, 51.3% had lesions somewhere and between the 38 analyzed after maceration, 73.7% also had injuries. 45% of the lesions from the 39 heads were in the maxilla, 15% in the mandible and 40% in both structures (maxilla and mandible) and from those heads with injuries observed after maceration 50% of the lesions were in the maxilla and 50% in the maxilla and mandible. Between the 17 heads of adult animals analyzed in the post-mortem examination and after maceration all had lesions somewhere. In the post-mortem examination, 11.8% had lesions in the mandible and 88.2% in both structures; after maceration, 5.9% were in the maxilla, 11.8% in the mandible and 82.3% in both structures. The histopathological findings revealed a chronic pyogranulomatous inflammatory process. To characterize the subgingival microbiota of the 14 sheep with periodontitis it was performed the Polymerase Chain Reaction (PCR), especially for microorganisms belonging to the red complex Socransky (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola) and other possible Gram-negative and Gram-positive periodontopathogens. There were identified in 50% of the samples Porphyromonas gingivalis, Tannerella forsythia in 92.8% and Treponema denticola in 78.5%. The three periodontal pathogens belonging to the red complex occurred simultaneously in 42.8% of the samples. We also identified in at least one of the examined animals Campylobacter rectus, Eikenella corrodens, Enterococcus faecium, Fusobacterium nucleatum, Prevotella intermedia, Prevotella nigrescens, and Prevotella loeschii. In the 14 samples Aggregatibacter actinomycetemcomitans, Dialister pneumosintes, Enterococcus faecalis and Porphyromonas gulae were not detected. The results showed that the sheep periodontitis occurred in the municipality of Benevides - PA has an infectious etiology and rushed a significant number of animals, many with bulging of the jaw; had high incidence in young and involved all of the adult animals post-mortem and after maceration examined.

Porphyromonas gingivalis, a Gram negative anaerobe implicated in the progression of periodontal disease, is capable of surviving and causing infection despite high levels of reactive nitrogen species found in the oral cavity due to its efficient nitrosative stress response. HcpR is an important sensor-regulator that plays a vital step in the initiation of the nitrosative stress response in many Gram negative anaerobic bacteria. We employ a combination of X-ray crystallography, SAXS, resonance Raman spectroscopy, UV-Vis spectroscopy, and molecular biology techniques to better understand this key regulator. Knockout of the hcpR gene in W83 P. gingivalis results in the inability of the bacteria to grow in physiological concentrations of nitrite and complementation of hcpR using the novel plasmid Pg108 rescues this phenotype. HcpR causes a drastic, dose dependent upregulation of PG0893, a gene coding for a putative NO reductase, when exposed to nitrite or nitric oxide. Full transcriptome sequencing reveals that hcp is the only significantly upregulated gene when P. gingivalis is exposed to nitrite and knockout of hcp resulted in a phenotype that is similar to that of the hcpR deficient strain. HcpR directly regulates the expression of hcp via direct binding to an inverted repeat sequence in the promoter region of the hcp gene. We present a 2.6 Å crystal structure of the N-terminal sensing domain of HcpR and show that it is FNR-CRP regulator. A putative hydrophobic heme binding pocket was identified in the junction between the N-terminal domain and the dimerization helix. Mutation of two methionine residues (Met68 and Met145) in this pocket abrogates activation of HcpR thus verifying the binding site. Heme bound to HcpR exhibits heme iron as a hexa-coordinate system in the absence of nitric oxide (NO) and upon nitrosylation transitions to a penta-coordinated system. Finally, Small Angle X-ray Scattering experiments of the full length HcpR reveal that the C-terminal DNA binding domain of HcpR has a high degree of interdomain flexibility.

Made available in DSpace on 2014-06-11T19:27:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-26Bitstream added on 2014-06-13T20:36:12Z : No. of bitstreams: 1 bedran_tbl_me_arafo.pdf: 840586 bytes, checksum: be6bd6ce48bcc11cac07694e0d0ef66c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A correta distinção dos micro-organismos envolvidos na patogênese da doença periodontal torna-se importante para o entendimento da sua progressão, auxiliando no adequado planejamento do tratamento periodontal. Assim métodos moleculares tornam-se ferramentas de grande valia no diagnóstico microbiológico. A técnica do PCR convencional é um método já estabelecido na literatura, porém não permite uma quantificação exata das amostras avaliadas. Com isso o PCR em tempo real surgiu para complementar o PCR convencional. O objetivo do presente estudo foi realizar a análise comparativa de ambas as técnicas na detecção de Porphyromonas gingivalis, Tannerella forsythia e Porhyromonas endodontalis. Foram selecionados 20 pacientes sistemicamente saudáveis, com doença periodontal crônica generalizada. Após o exame clínico periodontal completo, 30 amostras de sítios doentes não adjacentes (PS ≥ 5 e 7 mm com sangramento a sondagem) e 30 amostras de sítios sadios não adjacentes (PS ≤ 3 mm e ausência de sangramento a sondagem) foram coletados antes e 60 dias após a terapia periodontal básica de raspagem, alisamento radicular e instrução de higiene oral. O PCR convencional, PCR em tempo real e primers específicos para cada técnica foram utilizados para análise microbiológica. Para a análise comparativa entre as técnicas o PCR em tempo real foi dividido em 3 scores de acordo com a quantidade de DNA e o PCR convencional em 2 scores, presente e ausente. As análise estatísticas foram realizadas utilizados o software GraphPad. Os resultadosmostraram que o PCR em tempo real possui maior sensibilidade e especificidade na detecção dos micro-organismos P. endodontalis, P. gingivalis e T. forsythia, os quais foram detectados em maiores proporções nos sítios doentes quando comparados aos sítios... / The correct distinguishment of microorganisms involved in the periodontal disease pathogen, it is important in the understanding of its progression and adequate treatment planning, in this way, different microbiologic diagnostic molecular methods became key instruments. The conventional PCR technique is a well established method, however does not permit a precise quantification of the evaluated samples, being complemented by the real time PCR. The aim of the present study was to realize a comparative analysis of both techniques in the detection of Porphyromonas gingivalis, Tannerella forsythia e Porhyromonas endodontalis. 20 systemically healthy patients, with established generalized cronic periodontal disease were selected. After a complete clinical periodontal exam, 30 non adjacent affected sites samples (Depth Probing ≥ 5 e 7 mm with positive bleeding) and 30 non adjacent healthy sites samples (depth probing ≤ 3 mm with negative bleeding) were collected initially and 60 days after a basic periodontal therapy, root scaling and oral hygiene instructions. Conventional PCR, real time PCR and specific primers for it technique were used for microbiological analysis. To permit a comparative analysis between both PCR methods studied, the real time PCR was divided in 3 different scores, in accordance to DNA quantity and the conventional PCR in 2 scores, present or absent. The GraphPad software was used to make the statistical analysis. The results demonstrated that the real time PCR are more sensitive and specific in the detection of P. endodontalis, P. gingivalis and T. forsythia, detecting them in a higher proportion in the affected sites when compared to the healthy sites. After the basic periodontal therapy, there was a significant reduction of the bacteria analyzed. That way, can be suggested that presence of... (Complete abstract, click electronic access below)

Resumo: A correta distinção dos micro-organismos envolvidos na patogênese da doença periodontal torna-se importante para o entendimento da sua progressão, auxiliando no adequado planejamento do tratamento periodontal. Assim métodos moleculares tornam-se ferramentas de grande valia no diagnóstico microbiológico. A técnica do PCR convencional é um método já estabelecido na literatura, porém não permite uma quantificação exata das amostras avaliadas. Com isso o PCR em tempo real surgiu para complementar o PCR convencional. O objetivo do presente estudo foi realizar a análise comparativa de ambas as técnicas na detecção de Porphyromonas gingivalis, Tannerella forsythia e Porhyromonas endodontalis. Foram selecionados 20 pacientes sistemicamente saudáveis, com doença periodontal crônica generalizada. Após o exame clínico periodontal completo, 30 amostras de sítios doentes não adjacentes (PS ≥ 5 e 7 mm com sangramento a sondagem) e 30 amostras de sítios sadios não adjacentes (PS ≤ 3 mm e ausência de sangramento a sondagem) foram coletados antes e 60 dias após a terapia periodontal básica de raspagem, alisamento radicular e instrução de higiene oral. O PCR convencional, PCR em tempo real e primers específicos para cada técnica foram utilizados para análise microbiológica. Para a análise comparativa entre as técnicas o PCR em tempo real foi dividido em 3 scores de acordo com a quantidade de DNA e o PCR convencional em 2 scores, presente e ausente. As análise estatísticas foram realizadas utilizados o software GraphPad. Os resultadosmostraram que o PCR em tempo real possui maior sensibilidade e especificidade na detecção dos micro-organismos P. endodontalis, P. gingivalis e T. forsythia, os quais foram detectados em maiores proporções nos sítios doentes quando comparados aos sítios... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The correct distinguishment of microorganisms involved in the periodontal disease pathogen, it is important in the understanding of its progression and adequate treatment planning, in this way, different microbiologic diagnostic molecular methods became key instruments. The conventional PCR technique is a well established method, however does not permit a precise quantification of the evaluated samples, being complemented by the real time PCR. The aim of the present study was to realize a comparative analysis of both techniques in the detection of Porphyromonas gingivalis, Tannerella forsythia e Porhyromonas endodontalis. 20 systemically healthy patients, with established generalized cronic periodontal disease were selected. After a complete clinical periodontal exam, 30 non adjacent affected sites samples (Depth Probing ≥ 5 e 7 mm with positive bleeding) and 30 non adjacent healthy sites samples (depth probing ≤ 3 mm with negative bleeding) were collected initially and 60 days after a basic periodontal therapy, root scaling and oral hygiene instructions. Conventional PCR, real time PCR and specific primers for it technique were used for microbiological analysis. To permit a comparative analysis between both PCR methods studied, the real time PCR was divided in 3 different scores, in accordance to DNA quantity and the conventional PCR in 2 scores, present or absent. The GraphPad software was used to make the statistical analysis. The results demonstrated that the real time PCR are more sensitive and specific in the detection of P. endodontalis, P. gingivalis and T. forsythia, detecting them in a higher proportion in the affected sites when compared to the healthy sites. After the basic periodontal therapy, there was a significant reduction of the bacteria analyzed. That way, can be suggested that presence of... (Complete abstract, click electronic access below) / Orientador: Denise Madalena Palomari Spolidorio / Coorientador: Rosemary Adriana Chiérici Marcantonio / Banca: Poliana Duarte / Banca: Joni Augusto Cirelli / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / Endodontic treatment of the infected immature tooth has undergone a dramatic change. Conventional endodontic treatment can control infection, but root development usually remains impaired. A novel regenerative endodontic procedure, the revascularization method, can now control the infection and enable such teeth to continue root development. This is done by creating a fibrin-matrix scaffold in the antibiotic treated root canal space (RCS). Dental stem cells and growth factors have been able to continue root development in such an environment. The fibrin-matrix scaffold is dependent on the induction of a blood clot into the RCS, and this cannot always be predictably induced. PDS is a biocompatible material that can be electrospun to provide a matrix for cells and growth factors and perhaps improve on the blood clot induced fibrin scaffold by incorporating metronidazole as an adjuvant antimicrobial. A metronidazole containing electrospun PDS scaffold was examined in vitro using a turbidimetric test, the modified direct contact test. This scaffold significantly inhibited growth of an anaerobic primary endodontic pathogen Porphyromonas gingivalis. This scaffold may improve the treatment of the infected immature tooth by providing a designed matrix for root regeneration while serving simultaneously as an antibiotic drug delivery device to disinfect the RCS. The aim of this study is to evaluate in vitro the property of a synthetic scaffold to function as an antibacterial drug delivery device. PDS*II (polydioxanone) suture was obtained from Ethicon, INC. (Somerville, NJ) and was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol, HFIP (Sigma Aldrich). Three different scaffolds were electrospun onto an aluminum foil background; (1) control scaffold with no antibiotic incorporated, (2) scaffold with 5.0-wt % metronidazole incorporated, and (3) 25-wt % metronidazole incorporated. All scaffolds were cut using a 4-mm diameter biopsy punch under aseptic conditions and removed from foil, control scaffold (n = 64), scaffold containing 5.0-wt % metronidazole (n = 32), and scaffold containing 25-wt % metronidazole (n=32). Experimental scaffolds were placed in a 96- well sterile flat bottom microtiter plate. Porphyromonas gingivalis a known primary endodontic pathogen was grown in 5 ml of BHI + YE with 0.25 μl of vitamin K with incubation at 37°C under anaerobic conditions for 48 hours. Microplates were sterilized before inoculation with Pg with 400 μl of 70-percent EtOH for a minimum of 30 minutes then pipetted out. After sterilization the microwells were washed with 400 μl of sterile water and pipetted out. Group 1 (negative control) microwells (n = 8) contained control scaffold and 190 μl of broth only. Group 2 (positive control) microwells (n = 8) contained 190 μl of broth and Pg only. Group 3 microwells (n = 8) contained control scaffold, 190 μl of broth, and 10 μl of Pg inoculum. Group 4 microwells (n = 8) contained scaffold with 5 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 5 microwells (n = 8) contained scaffold with 25 wt % metronidazole, 190 μl of broth, and 10 μl of Pg inoculum. Group 6 contained 190 μl of uninoculated broth for spectrophotometer calibration. Sterile microplate lids were used to isolate microwells from the surrounding environment. Microplates were incubated at 37°C under anaerobic conditions for 48 hours. After 48 hours the microplates were read by using an endpoint reading in the spectrophotometer. This was repeated four times. Comparisons among the groups for differences in optical density as a measure of bacterial growth were made using mixed-model ANOVA, with a fixed effect for group and a random effect for experimental run. Pair-wise group comparisons were performed using Tukey's multiple comparisons procedure to control the overall significance level at 5 percent. The analyses were performed using the ranks of the data. Broth had significantly lower OD than all other groups (p < 0.0001). Broth+Pg and Broth+Pg+Scaffold had significantly higher OD than 5-wt % Metro (p < 0.0001) and 25-wt % Metro (p < 0.0001), but Broth+Pg and Broth+Pg+Scaffold were not significantly different from each other (p = 0.97). 5-wt % Metro and 25-wt % Metro were not significantly different from each other (p = 0.24). From the results of our study, we concluded that the 5.0-wt % and 25-wt % metronidazole containing scaffolds significantly inhibited bacterial growth and could be effectively utilized for the endodontic regeneration procedure.

No description available.

FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Over the past few years, there has been increasing evidence of the effect of the oral health over the general health of individuals, supported by a series of biological and epidemiological studies that show a relation between the mouth and many diseases, including cardiovascular diseases. Structural deficiencies and functional abnormalities of heart valves represent an important cause of cardiovascular morbidity and mortality in Brazil, and a few defects have been recently associated with infectious agents. The aim of this study was to identify cariogenic and periodontopathogenic bacteria in dental plaque, saliva and heart valves, without clinical endocarditis, of patients with heart valve diseases, and correlate these findings with the oral health status of the patients. Oral exams using the DMTF (decayed, missing and filled teeth) and PSR (Periodontal Screening and Recording) indexes to evaluate caries and periodontal disease, respectively, were performed. Samples of supragingival and subgingival dental plaque, saliva and cardiac valves were evaluated, through Real Time Polymerase Chain Reaction, for the presence of DNA of Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) and Treponema denticola (T. denticola). A total of 114 samples were collected from 42 patients with a mean age of 55.6  13.8 years. The average number of missing teeth due to caries was 23.52  9.41 teeth per patient, and according to the highest score of periodontal disease observed for each patient, excluding edentulous patients (44.0%), periodontal pockets over 4mm (43.4%) and dental calculus (34.7%) were detected in a higher number of patients. The molecular analysis of the oral samples revealed high frequency of S. mutans and P. intermedia in supragingival dental plaques, subgingival dental plaques and saliva of dentate and edentulous patients (variation 60.0% - 100.0%), while P. gingivalis and T. denticola were detected in a smaller number of oral samples (variation 17.6% - 64.0%). The microorganism most frequently detected in heart valve samples was the S. mutans (89.3%), followed by P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). Significant difference was observed between the frequency of P. intermedia, P. gingivalis and T. denticola in the heart valve and dental plaque, as oposed to S. mutans. The identification of oral bacteria, especially S. mutans, in heart valves of patients with a previous history of dental caries and gingivitis/periodontitis suggests the possible involvement of these pathogens in the etiopathogenesis of heart valve diseases. / Atualmente, cada vez mais se tem evidÃncias do efeito da condiÃÃo oral na saÃde geral dos indivÃduos, atravÃs de uma sÃrie de estudos epidemiolÃgicos e biolÃgicos que mostram uma relaÃÃo entre a boca e diversas doenÃas, incluindo as doenÃas cardiovasculares. Desordens estruturais e nas funÃÃes das vÃlvulas cardÃacas representam uma importante causa de morbidade e mortalidade cardiovascular no Brasil, sendo alguns processos, como a estenose aÃrtica degenerativa, mais recentemente associados a agentes infecciosos. O objetivo desta pesquisa foi identificar bactÃrias cariogÃnicas e periodontopatogÃnicas na placa dental, saliva e vÃlvulas cardÃacas, sem endocardite clÃnica, de pacientes com doenÃa valvar, correlacionando esses achados à condiÃÃo bucal dos indivÃduos. AvaliaÃÃo, quanto Ãs doenÃas cÃrie e periodontal, foi realizada, atravÃs dos Ãndices CPO-D (Dentes Permanentes Cariados, Perdidos e Obturados) e PSR (Registro Periodontal Simplificado), respectivamente. Amostras de placa dental supragengival, subgengival, saliva e vÃlvula cardÃaca foram coletadas para investigaÃÃo da presenÃa de DNA, atravÃs de PCR (ReaÃÃo em Cadeia de Polimerase) em tempo real, de Streptococcus mutans (S. mutans), Prevotella intermedia (P. intermedia), Porphyromonas gingivalis (P. gingivalis) e Treponema denticola (T. denticola). Um total de 114 amostras foi coletado de 42 pacientes com mÃdia de idade de 55.6  13.8 anos. A mÃdia de dentes perdidos devido à cÃrie, por paciente, foi em torno de 23.52  9.41 e, segundo o maior grau de doenÃa periodontal observado no indivÃduo, excluindo-se os pacientes desdentados totais (44.0%), bolsa superior a 4 mm (43.4%) e o cÃlculo dental (34.7%) esteve presente em um maior nÃmero de pacientes. A anÃlise molecular das amostras bucais revelou alta frequÃncia de S. mutans e P. intermedia nas placas supragengival, subgengival e saliva de pacientes dentados e desdentados (variando entre 60.0% e 100.0%), enquanto que P. gingivalis e T. denticola estiveram presentes em menor nÃmero de amostras bucais (variando entre 17.6% e 64.0%). O micro-organismo mais frequentemente encontrado nas amostras valvares foi o S. mutans (89.3%), seguido da P. intermedia (19.1%), P. gingivalis (4.2%) e T. denticola (2.1%). DiferenÃa significativa foi encontrada entre a presenÃa de P. intermedia, P. gingivalis e T. denticola na vÃlvula e na placa dental, diferentemente do S. mutans. A identificaÃÃo de bactÃrias orais, principalmente S. mutans, em vÃlvulas cardÃacas de pacientes com elevada experiÃncia prÃvia de cÃrie e ocorrÃncia de gengivite/periodontite, sugere o possÃvel envolvimento desses patÃgenos nas doenÃas valvares.

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells.

One of the greatest threats to human health is increasing antimicrobial resistance among pathogens, and finding alternatives for treatment of bacterial infections is of highest importance together with a more controlled use of antibiotics. Porphyrins and heme have both been shown to be a promising class of compounds for inactivation of bacteria; porphyrins by their excellent properties to act as a photosensitizer, and heme by its importance as an iron source during a bacterial infection in vertebrates. This thesis describes the development of analytical methods for the identification and determination of porphyrins and heme using liquid chromatography coupled to tandem mass spectrometry. Subsequently, these developed methods were applied to bacterial samples to investigate different culture conditions and additives effect to the intracellular porphyrin and heme composition. Singlet oxygen production of three naturally occurring porphyrins have been determined together with the photosensitivity for blue light and the porphyrin content in E. coli. Toothbrushes equipped with a LED, emitting light with a wavelength of 450 nm, were used in an eight week randomized clinical trial to investigate any positive periodontal effect of blue light. Porphyrin and heme content in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were highly affected by the different cultivation conditions. The culture age of A. actinomycetemcomitans affected the porphyrin profile, while only small changes were observed for P. gingivalis during growth. A large change of the porphyrin profile could be observed when the bacteria were passaged onto a new growth medium. Additional porphyrins were detected and the total porphyrin content increased up to 28 times. These findings highlight the need for more standardized cultivation procedures when performing in vitro experiments. Heme content in Escherichia coli was affected when different additives related to biosynthesis of heme were added to the growth medium. The uptake of heme could be reduced with 52% when a compound that chemically looks similar to heme was added to the growth medium. Since heme acquisition is important for many pathogens, this could be a promising target for antimicrobial drugs. E. coli showed no sensitivity for 405 nm light using light doses up to 172.8 J/cm2 and only low concentrations of porphyrins could be quantified. By adding a porphyrin precursor to E. coli the intracellular concentration of porphyrins increased remarkably and a light dose of 57.6 J/cm2 reduced the bacterial number with &gt; 5 log10 steps. This shows that E. coli can be killed due to their endogenous porphyrins. In the clinical study we could see a weak trend that the 450 nm LED toothbrush possessed a phototherapeutic effect for three clinical indices. All indices were decreased in the intervention group, but there were no statistically significant difference compared to the control group. However, four inflammation markers were significantly decreased in the intervention group while only one decreased significantly in the control group. In conclusion, this thesis has shown that porphyrins and heme are produced endogenously in microorganisms and that the porphyrin profiles vary depending on culture conditions and different additives. Furthermore, porphyrins may be used as endogenous photosensitizers to inactivate bacteria, but more research is necessary to determine if there is a specific porphyrin that contributes more to the photosensitivity. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

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