M.Sc. / LPS, as a pathogen associated molecular pattern (PAMP) molecule can interact with eukaryotic host cells. Interaction occurs by either direct contact or due to the release of micelles containing LPS from bacterial cell surfaces. LPS activates innate host defence systems in both invertebrate and vertebrate animal/insect cells via analogous pathways, where the lipid A component,is responsible for the activities. LPS from several plant pathogens have been shown to activate a number of defence-related responses in plants. Initial concentration studies and cell viability assays were conducted to assess isonitrosoacetophenone (INAP) and LPS as elicitors of defensive responses in tobacco (Nicotiana tabacum cv. Samsun) cell suspensions. The effective concentrations were found to be 100vM INAP and 100μg/ml LPS. RNA was isolated, quantified and analysed to confirm the quality of the starting material for differential display analysis. The DDRT-PCR technique was successfully applied in order to obtain comparative "displays" of PCR amplicons derived from three sub-divided mRNA pools (i.e. each of the three different anchor primers, per treatment). Significant differences in the profiles of control, INAP and LPS treated cells were observed, indicating that the eliciting agents had prominent effects on cellular homeostasis, resulting in an altered gene expression profile. DDRT-PCR can be technically challenging at a number of steps. Modifications were incorporated to initially obtain differentially expressed transcripts (DETs), as well as reamplify the DETs. 223 Putative DETs were isolated from denaturing polyacrylamide sequencing gels. 172 Putative DETs were re-amplified, of which 126 appeared as good candidates for further analysis. Finally, 96 putative DETs were chosen for reverse Northern analysis. DDRT-PCR has been reported to be plagued by false positives. Reverse Northern analysis confirms the presence of the putative DET from the subdivided RNA pool, as well as affirming the differential expression, compared between the control and inducer blots. 26 DETs were selected for cloning, of which 16 were sequenced. Homologies between the DETs and known sequences were determined using BLASTN and BLASTX alignments, DNAssist software, as well as MIPS alignments to the Arabidopsis genome. Five of the DETs were assigned putative functions in plant signal perception, transduction and the defence response, based on their respective sequence homologies to sequences involved in innate immunity. It is proposed that the DET, HAP3-15, represents the plant equivalent of a component of the innate immunity pathway in mammals and Drosophila. It is further proposed that HAP3-15 represents a S-Receptor kinase protein (SRK), with a defensive role in distinguishing self from potential pathogens. Therefore, as a SRK, HAP3-15 would function as a transmembrane receptor able to conduct an external signal through the membrane to the cytoplasm as a form of signal perception. Subsequently HAP3-15 could ii play a role in phosphorylation cascades through the kinase domain and, consequently, be responsible for signal transduction. In addition, LPS would then represent the ligand creating the signal perceived by the SRK, HAP3-15, with oligosaccharide binding ability. HAP3-15 was also identified as a true positive by the INAP probe in reverse Northerns, implying that both the biological and chemical inducers used, activated the same receptor kinase. Whether the same signalling pathway was followed during the phosphorylation cascades has not been determined. Further analysis will require Northern blots in a time study to investigate the kinetics of induction. In addition, longer sequence information for each of the five DETs needs to be obtained to identify the corresponding genes in order to investigate their roles in innate immunity in plants.

>Magister Scientiae - MSc / Genetic variability in genes encoding drug metabolizing enzymes, transporters and targets are known to be the main factors of inter-individual differences in therapeutic outcome. Genetic factors are estimated to be responsible for about 15-30% of inter-individual variation in drug disposition and response. Single-nucleotide polymorphisms (SNPs) are the most prevalent class of genetic variation that could explain the variability in drug efficacy and undesired side effects for patients. The aims of this study were to develop and evaluate the performance of robust and high throughput techniques for genotyping ten polymorphisms related to anticancer drugs and ten polymorphisms related to cholesterol lowering drugs. SNaPshot minisequencing and high resolution melt analysis (HRM) genotyping panels were developed, optimized, and their performances were evaluated and compared. SNaPshot minisequencing systems were developed and successfully optimized for the genotyping of ten SNPs associated with anticancer drug therapy, and ten SNPs associated with cholesterol lowering drugs. These systems were used to genotype the selected SNPs in 130 healthy Cape Admixed participants residing in Cape Town, South Africa. Population genetics data obtained for the studied SNPs were analysed using several statistical analysis software tools. Important population genetic parameters were calculated. Among others, allelic and genotypic frequencies were determined and compared with other populations in the world. High resolution melt analysis (HRM) genotyping panels were developed, optimized and their performance were evaluated and compared to the SNaPshot assays. HRM was explored as an alternative inexpensive and rapid methodology to genotype five SNPs related to anticancer therapy and five SNPs related to cholesterol lowering therapy (statins). Unlike the SNaPshot assays, rigorous optimization was required for the detection heterozygous genotypes via HRM. Both assays were validated using direct sequencing and compared to each other. The HRM system is a closed tube, cheap and (theoretically) rapid method for identifying genetic variations. HRM was however found to be more time consuming, needed further optimization, primer redesigning and more evaluation. The developed genotyping systems could be further validated using clinical samples from patients. This could help in optimizing drug therapy for cancer and cholesterol treatment.

South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.

The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.

Human cytomegalovirus (HCMV) infection is the major cause of illness and death in immunocompromised patients. HCMV is the most common cause of congenital viral infection in humans. A polymerase chain reaction (PCR) method was developed for the rapid detection of CMV in urine. Several parameters of the PCR procedure were optimized to reduce time and improve sensitivity. By eliminating the extraction of DNA from clinical specimens, reducing the number of amplification cycles, utilization of the "hot start" PCR procedure and direct detection of PCR product by ethidium bromide fluorescence staining, a procedure was developed which could be performed in less than 3 hours. Comparison studies using cell culture and direct detection of CMV by PCR on urine specimens were performed. Sensitivity was further examined to determine if inhibitors of the PCR reaction were present in urine.

The broader research area investigated during this programme was conceived from a goal to contribute towards solving the challenge of food security in the 21st century through the reduction of crop loss and minimisation of fungicide use. This is aimed to be achieved through the introduction of an empirical approach to agricultural disease monitoring. In line with this, the SYIELD project, initiated by a consortium involving University of Manchester and Syngenta, among others, proposed a novel biosensor design that can electrochemically detect viable airborne pathogens by exploiting the biology of plant-pathogen interaction. This approach offers improvement on the inefficient and largely experimental methods currently used. Within this context, this PhD focused on the adoption of multidisciplinary methods to address three key objectives that are central to the success of the SYIELD project: local spore ingress near canopies, the evaluation of a suitable model that can describe spore transport, and multivariate analysis of the potential monitoring network built from these biosensors. The local transport of spores was first investigated by carrying out a field trial experiment at Rothamsted Research UK in order to investigate spore ingress in OSR canopies, generate reliable data for testing the prototype biosensor, and evaluate a trajectory model. During the experiment, spores were air-sampled and quantified using established manual detection methods. Results showed that the manual methods, such as colourimetric detection are more sensitive than the proposed biosensor, suggesting the proxy measurement mechanism used by the biosensor may not be reliable in live deployments where spores are likely to be contaminated by impurities and other inhibitors of oxalic acid production. Spores quantified using the more reliable quantitative Polymerase Chain Reaction proved informative and provided novel of data of high experimental value. The dispersal of this data was found to fit a power decay law, a finding that is consistent with experiments in other crops. In the second area investigated, a 3D backward Lagrangian Stochastic model was parameterised and evaluated with the field trial data. The bLS model, parameterised with Monin-Obukhov Similarity Theory (MOST) variables showed good agreement with experimental data and compared favourably in terms of performance statistics with a recent application of an LS model in a maize canopy. Results obtained from the model were found to be more accurate above the canopy than below it. This was attributed to a higher error during initialisation of release velocities below the canopy. Overall, the bLS model performed well and demonstrated suitability for adoption in estimating above-canopy spore concentration profiles which can further be used for designing efficient deployment strategies. The final area of focus was the monitoring of a potential biosensor network. A novel framework based on Multivariate Statistical Process Control concepts was proposed and applied to data from a pollution-monitoring network. The main limitation of traditional MSPC in spatial data applications was identified as a lack of spatial awareness by the PCA model when considering correlation breakdowns caused by an incoming erroneous observation. This resulted in misclassification of healthy measurements as erroneous. The proposed Kriging-augmented MSPC approach was able to incorporate this capability and significantly reduce the number of false alarms.

Orientador: Regina Maura Bueno Franco / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T02:37:18Z (GMT). No. of bitstreams: 1 Leal_DiegoAveraldoGuiguet_D.pdf: 4157800 bytes, checksum: 6b17942eb61d5ff1784bbd719d71b15b (MD5) Previous issue date: 2013 / Resumo: Surtos de doenças gastrointestinais associados com a ingestão de moluscos bivalves crus ou mal cozidos têm sido reportados em todo o mundo. O procedimento mais utilizado para purificar os tecidos dos bivalves, consiste na utilização de água desinfetada com radiação UV aplicada em tanques de depuração, previamente à introdução dos animais no mercado. Os riscos de aquisição de infecções mediante o consumo de bivalves são maximizados quando estes são cultivados em áreas inapropriadas, ou quando são comercializados sem serem submetidos ao procedimento de depuração. Os objetivos deste estudo foram: avaliar a contaminação por cistos de Giardia duodenalis e oocistos de Cryptosporidium em ostras destinadas ao consumo humano, antes e após o procedimento de depuração com luz UV, e em águas salobras, nas etapas relacionadas com a cadeia produtiva de ostras no estuário de Cananéia, litoral sul de São Paulo; realizar a caracterização molecular de cistos de Giardia em diferentes etapas da produção de ostras para verificar os genótipos circulantes e aqueles relacionados com a infecção em humanos; enumerar a concentração de indicadores bacteriológicos de contaminação fecal, da área de cultivo até a etapa de depuração para verificar se atende à regulamentação do Conselho Nacional do Meio Ambiente (CONAMA). Quatro pontos foram selecionados para coleta de água e ostras e pesquisa de protozoários: ponto 1: região de cultivo e engorda das ostras; ponto 2: captação de água; ponto 3: etapa de filtração de água e ponto 4: tanque de depuração. As amostras de água foram analisadas por filtração em membranas seguida de separação imunomagnética (IMS) e reação de imunofluorescência direta (RID). A contaminação das ostras foi verificada mediante análise de conteúdo líquido interno e lavado branquial por IMS e RID. A reação em cadeia da polimerase e o sequenciamento foram realizados para pesquisa de Giardia duodenalis e caracterização de seus genótipos. Giardia foi o patógeno mais prevalente em todos os pontos de água, sendo detectada em: 50,0%, 25,0%, 33,3% e 50,0% das amostras dos pontos 1, 2, 3 e 4, respectivamente. O subgenótipo AII foi detectado em algumas amostras hídricas dos pontos 1, 2 e 4 e o genótipo C em duas amostras (pontos 3 e 4). Oocistos de Cryptosporidium foram detectados em 16,6% das amostras de água após a depuração. As ostras da região de cultivo continham cistos de Giardia duodenalis em 41,6% das amostras; o subgenótipo AII foi evidenciado em uma amostra. As ostras depuradas apresentaram a maior contaminação: 58,3% das amostras albergavam cistos de Giardia e 3 delas pertenciam ao subgenótipo AII. As análises bacteriológicas revelaram que o ponto de engorda (P1) estava adequado para o cultivo das ostras. Estes resultados refletem a ineficiência do procedimento de depuração com UV aplicado nesta planta de tratamento e a contaminação do produto - na etapa final que precede sua comercialização - por Giardia duodenalis pertencente ao subgenótipo AII, potencialmente infectante para seres humanos. Os resultados evidenciam a necessidade de monitoramento contínuo de patógenos e adequação da legislação do cultivo de moluscos bivalves no Brasil, a fim de garantir a segurança alimentar / Abstract: Many shellfish-borne associated outbreaks have been reported worldwide especially related to its ingestion raw or lightly cooked. In the shellfish industry, the most commonly utilized procedure to purify shellfish tissues consists in the utilization of UV disinfected water employed at depuration tanks, before they are placed on the market. The risks of acquiring gastroenteric diseases, through its consumption, are maximized when they are cultured on inappropriate growing areas or whenever they are sold without depuration procedure. The purposes of this study were: evaluate the contamination by Giardia duodenalis cysts and Cryptosporidium oocysts in oysters destined to human consumption, before and after UV depuration procedure, and in brackish waters at stages related to the food chain of edible oysters, from Cananéia estuary located on the south coast of São Paulo; perform molecular characterization of Giardia cysts at different stages of the production of oysters to verify the main circulating genotypes and those associated with human infection; enumerate bacteriological indicators of fecal contamination from growing site up to depuration site, to check if it complies with the National Environmental Council (CONAMA). Four sampling sites were selected for water and oysters harvesting and protozoa search: site 1- oysters growing area; site 2: catchment water; site 3: filtration stage of water treatment and site 4: oyster's depuration tank. Water samples were analyzed through membrane filtration technique followed by immunomagnetic separation (IMS) and direct immunofluorescence assay (IFA). Oysters' contamination was verified through the analysis of innerwater and gill wash using IMS and IFA. The polymerase chain reaction and sequencing reactions were performed to search for Giardia duodenalis and characterization of its genotypes. Giardia was the most prevalent pathogens in all water sites, where it was detected: 50.0%, 25.0%, 33.3% and 50.0% of water from sites 1, 2, 3 and 4 respectively. The subgenotype AII was detected in several water samples from sites 1, 2 and 4 and the genotype C in two samples (sites 3 and 4). Cryptosporidium oocysts were found in 16.6% of depuration water tank. Oysters from growing area harbored Giardia duodenalis cysts in 41.6% of the samples and subgenotype AII was evidenced once. Depurated oysters were the most contaminated: 58.3% were harboring Giardia duodenalis cysts and 3 belonged to subgenotype AII. Bacteriological analysis demonstrated that site 1 was adequate to growing oysters. These results indicate the ineffectiveness of the UV depuration applied in this shellfish treatment plant, and the contamination of the product - in the final stage which precedes its commercialization - by Giardia duodenalis belonging to subgenotype AII, potentially infectious to humans. The results highlight the need for continued monitoring of pathogens and suit the legislation on the cultivation of bivalve molluscs in Brazil in order to ensure food safety / Doutorado / Parasitologia / Doutor em Parasitologia

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T10:59:39Z (GMT). No. of bitstreams: 1 Martinho_FredericoCanato_D.pdf: 10102770 bytes, checksum: 1acabb89bf73a5204986aa39e6e8213d (MD5) Previous issue date: 2011 / Resumo: Bactérias Gram-negativas (BG-) e seu sub-produto [Lipopolisacarídeo (LPS) - endotoxina) são capazes de estimular células a produzirem citocinas pró-inflamatórias envolvidas na destruíção tecidual periapical. Os objetivos propostos foram: 1) analisar os diferentes métodos de LAL para quantificação de endotoxinas, revelando o (s) que melhor (es) se adapta (m) para investigação de endotoxina nas infecções de origem endodôntica (capítulo 1); 2) estudar o perfil da microbiota e níveis de endotoxinas nas infecções endodônticas primárias com lesão periapical, determinando a antigenicidade do conteúdo endodôntico contra macrófagos através da produção de IL1- ß e TNF-? (capítulo 2); 3) investigar a presença de espécies bacterianas Gram-negativas "alvos" e níveis de endotoxinas nas infecções endodônticas primárias com lesão periapical, determinando seu potencial antigênico contra macrófagos através da produção de PGE2 (capítulo 3); 4) detectar espécies de Treponema spp e os níveis de endotoxinas em infecções endodônticas primárias e determinar sua antigenicidade contra macrófagos através dos níveis de IL-6 e IL-10, avaliando sua correlação com os achados clínicos e radiográficos (capítulo 4); 5) avaliar a atividade antigênica de LPS isolado de P. gingivalis e F. nucleatum encontrados em canais radiculares infectados sobre macrófagos (RAW 264.7) através dos níveis de IL-1? e TNF-? (capítulo 5); 6) comparar "in vivo" a efetividade do preparo químico-mecânico com NaOCl 2.5% e CLX-gel 2% na eliminação de LPS de bactérias orais presentes em dentes com necrose pulpar e presença de lesão periapical (capítulo 6); 7) avaliar o efeito do preparo químico-mecânico com NaOCl 2.5% + EDTA 17% e limas rotatórias NiTi (Mtwo®) na remoção de endotoxinas de dentes com necrose pulpar e presença de lesão periapical (capítulo 7); 8) comparar a capacidade de diferentes sequências clínicas do sistema rotatório Mtwo® na remoção de endotoxinas em canais radiculares contaminados. (capítulo 8). Método: amostras foram coletadas de canais radiculares com IEPL utilizando cones de papel estéreis/despirogenizados. PCR (16s rDNA) e método LAL foram utilizados. Níveis de citocinas inflamatórias foram quantificados através de ELISA (Duoset-Kit, R&D systems). Resultados: Os testes cinéticos (KQCL - Kinetic Quantitative Chromegenic Limulus e turbidimétrico) mostraram níveis de endotoxinas inferiores (7,49 EU/mL e 9,19 EU/mL, respectivamente), quando comparados ao teste QCL (Quantitative Chromegenic Limulus) (34,20 EU/mL) (p<0,05). Prevotella nigrescens (13/21) foi mais frequentemente encontrada. Dente com exsudato foi relacionado com a presença de F. alocis (p<0,05). Correlações positivas (p<0,05) foram encontradas entre: número de BG- e níveis de IL-1 ß, TNF-? e PGE2; níveis de endotoxinas e de TNF-? (p<0,05); IL-1 ß e tamanho de lesão periapical. Endotoxina foi detectada em 100% dos canais radiculares estudados. Maior redução de entodotoxina foi encontrada nos dentes instrumentados com NaOCl 2,5% (57,98%) versus CLX-gel 2% (47,12%) (p<0,05) utilizando limas manuais K-file. Após PQM com NaOCl 2,5% e limas rotatórias NiTi endotoxina foi reduzida em 98,06% (p<0,05). Conclusão: 1) Os testes cinéticos turbidimétrico e cromogênico de LAL apresentaram resultados mais precisos e de melhor reprodutibilidade quando comparados ao QCL (capítulo 1);. 2) A antigenicidade do conteúdo endodôntico não está relacionada apenas com a quantidade de endotoxinas encontrada nos canais radiculares, mas também com o número de diferentes espécies Gram-negativas presentes na infecção. Maior destruíção óssea periapical foi relacionado com níveis elevados de IL-1ß (capítulo 2); 3) O número de espécies BG- presentes nas IEPL foi relacionado com diferentes níveis de secreção de PGE2 via macrófagos. Maior produção de PGE2 foi relacionada com a presença de sintomatologia clínica (capítulo 3); 4) espécies de Treponema spp. exercem seu papel na patogênese das infecções endodônticas primárias. Além disso, o conteúdo bacteriano e particularmente os níveis de endotoxinas presents nos canais radiculares estimularam a produção de IL-6 e IL-10 por macrófagos (capítulo 4); 5) LPS isolados de P. gingivalis e F. nucleatum de canais radiculares infectados estimulam a produção de IL-1? e TNF-?, que são mediadores inflamatórios pleiotrópicos, podendo iniciar a resposta inflamatória e estimular a produção de mediadores secundários envolvidos na destruição tecidual (capítulo 5); 6) PQM com NaOCl 2,5% ou CLX-gel 2% não foram eficazes na eliminação de endotoxinas presentes na IEPL (capítulo 6); 7) PQM com NaOCl 2,5% + EDTA 17% e limas rotatórias NiTi (Mtwo®) foi eficaz na remoção de endotoxinas em 98,06% (capítulo 7); 8) redução significativa de endotoxinas foi obtida utilizando as sequências Mtwo® finalizadas com o preparo apical final #40.04 or #25.07 (capítulo 8) / Abstract: Gram-negative bacteria (G-ve) and its by-product [Lipopolysaccharide (LPS) - endotoxin] are capable to stimulate cells to release proinflammatory cytokines that lead to tissue destruction. The aims of this study were: 1) to determine which of the quantitative methods, namely, chromogenic endpoint, chromogenic kinetic and turbidimetric kinetic ones, best fit for the analysis of primary endodontic infections (chapter 1); 2) to investigate the microbial profile and the levels of endotoxin found in primary root canal infection with apical periodontitis (PEIAP), and to determine their antigenicity against macrophages through the levels of IL-1ß and TNF-alpha, evaluating their relationship with clinical and radiographic findings (chapter 2); 3) to investigate target G-ve bacteria species and endotoxin in PEIAP, determining their antigenicity against macrophages through the levels of PGE2 and evaluated their relationship with clinical findings (chapter 3); 4) investigation of Treponema spp. and endotoxin in primary endodontic infection and evaluation of the antigenicity of the infectious content against RAW 264.7 macrophages by the levels of IL-6 and IL-10 production (chapter 4); 5) to evaluate the antigenic activity of LPS purified from P. gingivalis and F. nulceatum isolated from infected root canals on macrophages cells (RAW 264.7) by the levels of IL-1? and TNF-?. (chapter 5); 6) to compare the efficacy of chemomechanical preparation with 2.5% NaOCl and 2% CHX-gel on eliminating oral bacterial LPS in teeth with PEIAP (chapter 6); 7) to investigate the ability of chemo-mechanical preparation (CMP) with 2.5% NaOCl + 17% EDTA and rotary NiTi system Mtwo® in removing endotoxin from PEIAP (chapter 7); 8) Comparison of different clinical sequences of NiTi rotary files Mtwo® in the removal of endotoxin from infected root canals (chapter 8). Methods: Samples were taken from root canals with PEIAP with paper points. PCR technique (16S rDNA) was used for the detection of the target bacteria. Limulus Amebocyte Lysate (LAL) was used to measure endotoxin. The amounts of IL-1ß, TNF-alpha and PGE2 in macrophages supernatants were measured by enzyme-linked immunosorbent assay - Duoset-kit (ELISA). Results: The KQCL and Turbidimetric -assay yielded a median value of endotoxin of 7.49 EU/mL and 9.19 EU/mL respectively, significantly different from the endpoint-QCL (34.20 EU/mL) (p<0.05). Prevotella nigrescens (13/21) was the most frequently Gram-negative bacteria species detected. Tooth with radiolucent area ? 2mm was related to Treponema denticola. Correlation was found between the number of Gram-negative bacteria and the levels of IL-1ß, TNF-alpha and PGE2 (p<0.05). Increased levels of endotoxin were followed by TNF-alpha release (p<0.05). Higher levels of IL-1ß (p<0.05) and endotoxin contents were related to the larger size of radiolucent area. Elevated levels of PGE2 were found in teeth with tenderness to percussion and pain on palpation. Endotoxin was detected in 100% of the root canals investigated. Higher percentage value of endotoxin reduction was found in 2.5% NaOCl (57.98%) when compared to 2% CHX-gel (47.12%) (p<0.05) using manual K-files. After chemo-mechanical preparation with 2.5% NaOCl and rotary NI-TI files endotoxin was significantly reduced to 98.06% (p<0.05). Conclusion: 1) Quantitative kinetic-turbidimetric and kinetic-chromogenic LAL methods are best fitted for analysis of endotoxin in root canal infection, both being more precise and allowing better reproducibility compared to the endpoint-QCL assay; 2) The antigenicity of the endodontic contents is not related to only the amount of endotoxin found in root canal, but also with the number of different species of Gram-negative bacteria involved in the infection. Moreover larger size (? 2mm) of radiolucent area was related to IL-1ß and endotoxin; 3) Additive effect between the number of G-ve bacterial species involved in endodontic infection regarding the induction of pro-inflammatory cytokine by macrophage cell. Moreover, teeth with clinical symptomatology were related to higher levels of endotoxin and PGE2 secretion; 4) a wide variety of Treponema species do play a role in primary endodontic. Moreover, the bacterial endodontic contents, particularly the levels of endotoxin present in root canals, were a potent stimuli for the production IL-6 and IL-10 in macrophages; 5) LPS of the P. gingivalis and F. nucleatum isolated from root canal infection is involved in the induction of IL-1 ? and TNF-?, which are pleiotropic inflammatory mediators, that can play a role in the initiation of the upregulation of the inflammatory response and can also stimulate the production of secondary mediators involved in tissue destruction; 6) 2.5% NaOCl and 2% CHX-gel were not effective on eliminating endotoxin from the primarily infected root canals using manual K-files; 7) CMP with 2.5% NaOCl + 17% EDTA and rotary NiTi files was effective in reducing 98.06% of endotoxin from PEIAP; 8) substantial reduction of endotoxin contents was achieved by using the Mtwo® sequences finished in APS #40.04 or #25.07 / Doutorado / Endodontia / Doutor em Clínica Odontológica

Orientador: Brenda Paula Figueiredo de Almeida Gomes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-18T14:01:31Z (GMT). No. of bitstreams: 1 Chiesa_WandersonMiguelMaia_D.pdf: 2308331 bytes, checksum: 52ef248e0f498c63a519acd9b0b14e9a (MD5) Previous issue date: 2011 / Resumo: Conteúdos dos canais radiculares podem ficar aderidos às limas endodônticas durante o preparo químico-mecânico, tais como endotoxinas, com grande potencial de desencadear resposta inflamatória. Instrumentos endodônticos são frequentemente reutilizados, existindo preocupação sobre a neutralização daqueles conteúdos pelos métodos de esterilização empregados em consultórios odontológicos. Os objetivos deste estudo foram: avaliar os efeitos de esterilização por estufa de calor seco ou autoclave, na detecção de DNA bacteriano e níveis de endotoxinas de limas endodônticas contaminadas in vivo, verificando correlações entre as espécies detectadas; investigar a produção de IL-1ß por macrófagos murinos estimulados in vitro por amostras coletadas de hastes metálicas contaminadas de limas endodônticas e esterilizadas por estufa de calor seco ou autoclave. Oitenta limas endodônticas manuais número 15 de aço inoxidável ficaram estéreis e apirogênicas, após esterilização pelo calor seco a 200ºC/4h. Vinte limas tiveram seus cabos removidos e as hastes metálicas destinadas ao Grupo I (Controle negativo), em frascos apirogênicos. Vinte pacientes apresentando necrose da polpa dental, lesão periapical e sem dor espontânea foram incluídos nesta pesquisa. Cavidades de acesso foram assepticamente preparadas e três limas apirogênicas sucessivamente introduzidas e removidas até o comprimento de trabalho de cada canal. Depois do uso, os cabos foram removidos, as hastes metálicas inseridas em frascos estéreis e apirogênicos e os outros grupos foram divididos (n=20): Grupo II (Controle positivo) - amostras sem esterilização; Group III - esterilização pela estufa de calor seco (170ºC/1h); Grupo IV - amostras autoclavadas. A técnica PCR (16S rDNA) foi usada para detectar DNA bacteriano, um teste turbidimétrico do Lisado dos Amebócitos de Limulus (LAL) foi usado para mensurar os níveis de endotoxinas e o método ELISA calculou as quantidades de IL-1ß liberadas por macrófagos murinos estimulados pelos conteúdos das limas contaminadas e esterilizadas. Prevotella nigrescens, Porphyromonas endodontalis e Treponema socranskii foram as bactérias-alvo mais frequentemente detectadas, a partir das amostras contaminadas e não esterilizadas. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia e Prevotella tannerae não foram identificadas. Correlação positiva estatisticamente significante (p<0.05) foi encontrada entre Porphyromonas endodontalis e Treponema denticola, e desta com Treponema socranskii. Depois de esterilização pela estufa de calor seco ou autoclave, o método PCR não detectou bactérias. O teste turbidimétrico indicou a mediana de 1,070 UE/mL, 0,875 UE/mL e 0,251 EU/mL, no grupo sem esterilização, esterilizado pela estufa de calor seco e autoclavado, respectivamente. Macrófagos estimulados pelas amostras das limas contaminadas e autoclavadas liberaram IL-1ß (mediana de 55,39 pg/mL). Conclusão: DNA bacteriano não foi detectado depois da esterilização pela estufa de calor seco ou autoclave, mas estes métodos não foram capazes de eliminar endotoxinas de limas endodônticas contaminadas e não foram encontradas diferenças estatisticamente significantes entre os dois métodos de esterilização empregados (p>0.05). Correlações positivas significantes foram detectadas entre micro-organismos colhidos de limas endodônticas contaminadas. Amostras autoclavadas estimularam a liberação de IL-1ß por macrófagos, mas não foram detectados níveis mensuráveis desta citocina depois da estimulação pelas amostras esterilizadas por calor seco / Abstract: Contents from root canal can be attached on endodontic files during chemomecanical preparation, such as endotoxin, with great potential to trigger inflammation. Endodontic instruments are often reused, and there is a concern about the neutralization of those contents by sterilization methods employed in dental offices. The objectives of this study were: to evaluate the effect of sterilization by dry heat oven or autoclave, in the bacterial DNA detection and endotoxin levels from in vivo contaminated endodontic files, verifying correlations between detected species; to investigate IL-1ß production by murine macrophage stimulated in vitro by samples from contaminated metal shafts of endodontic files and sterilized by dry heat oven or autoclave. Eighty size 15 stainless steel hand files were sterile and apyrogenic, after dry heat sterilization at 200ºC/4h. Twenty files had their handles cut off and metal shafts destined to the Group I (Negative control), in apyrogenic vials. Twenty patients presenting dental pulp necrosis, periapical lesion and without spontaneous pain were included in this research. Access cavities were aseptically prepared and three apyrogenic files were successively introduced and removed at the working length of each canal. After use, handles were cut off, metal shafts inserted in apyrogenic vials and the other groups were divided (n=20): Group II (Positive control) - samples with no sterilization; Group III - sterilization by dry heat oven (170ºC/1h); Group IV- autoclaved samples. PCR technique (16S rDNA) was used to detect bacterial DNA, Limulus Amebocyte Lysate (LAL) turbidimetric test measured endotoxin levels and ELISA method calculated amounts of IL-1ß released by murine macrophage stimulated by contents from the contaminated and sterilized files. Prevotella nigrescens, Porphyromonas endodontalis and Treponema socranskii were the most frequently detected target bacteria, from contaminated and non sterilized samples. Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tanerella forsythia and Prevotella tannerae were not identified. Statistically significant positive correlation (p<0.05) was found between Porphyromonas endodontalis and Treponema denticola, and between Treponema denticola and Treponema socranskii. After sterilization by dry heat oven or autoclave, PCR method detected no bacteria. Turbidimetric test indicated median of 1.070 EU/mL, 0.875 EU/mL and 0.251 EU/mL, in the group without sterilization, sterilized by dry heat oven and autoclaved, respectively. Macrophage stimulated by samples from contaminated and autoclaved files released IL-1ß (median of 55.39 pg/mL). Conclusion: no bacterial DNA was detected (PCR technique - 16S rDNA) after sterilization by dry heat oven or autoclave, but these methods were not able to eliminate endotoxin from contaminated endodontic files and no statistically significant differences were found between the two methods of sterilization employed (p>0.05). Significant positive correlations were detected between microorganisms collected from contaminated endodontic files. Autoclaved samples stimulated IL- 1ß releasing by macrophage, but no measurable levels of this cytokine were detected after stimulation by dry heat sterilized samples / Doutorado / Endodontia / Doutor em Clínica Odontológica

Masters of Science / Retinoblastoma Binding Protein 6 (RBBP6) is a RING finger-containing protein which plays a critical role in the 3'-end processing of mRNA transcripts. It is a constituent of the human pre-mRNA processing complex but also interacts directly with core splicing-associated proteins. RBBP6 also interacts with both major tumour suppressor proteins p53 and pRb and is known to play a critical role in suppression of p53 during development, in cooperation with MDM2. Through its RING finger it interacts with the C-terminus of the oncogenic protein Y-Box Binding Protein 1 (YB-1) both in vitro and in vivo, catalysing its ubiquitination and degradation in the proteasome. YB-1 is closely associated with tumour progression, poor patient prognosis and chemotherapeutic resistance, making it a promising target for therapeutic intervention. Unpublished data from our laboratory suggests that RBBP6 is able to poly-ubiquitinate YB-1 in vitro, using UbcH1 as the ubiquitin- conjugating enzyme (E2). This study aims to identify RBBP6 RING protein-protein interactions involved in the down regulation of YB-1 by RBBP6. These interactions include the C-terminal fragment of YB-1 (substrate), MDM2 (E3) and UbcH1 (E2). The C-terminal fragment of YB-1, denoted YB-1₂₂₀₋₃₂₄, was successfully cloned and expressed in bacteria and demonstrated to interact directly with the RBBP6 RING finger domain in in vitro affinity pull down assays. This is in good agreement with our unpublished data that RBBP6 is able to ubiquitinate full length YB-1 as well as the YB-1₂₂₀₋₃₂₄ fragment. UbcH1 was successfully expressed and shown to interact directly with RBBP6 RING in in vitro affinity pull down assays. This is also in agreement with our data showing that RBBP6 is able to ubiquitinate YB-1 using UbcH1 as E2. ¹⁵N-labelled samples of RBBP6 RING was successfully expressed in bacteria and used to investigate the putative interaction with UbcH1 in NMR-based chemical shift perturbation assays. However no interaction was observed, possibly because the sample of UbcH1 was subsequently found using mass spectrometery to be partially degraded. GST-tagged RBBP6 RING was able to precipitate MDM2 from HeLa lysate. This extends previous reports that full length RBBP6 and MDM2 interact directly and play a role in the suppression of p53 during development. The result was validated by showing that GST-MDM2 was able to precipitate RBBP6 RING in in vitro. This study includes a side project which involved the cloning and expression of DWNN-GG. GST-HADWNN-GG was successfully cloned and expressed in bacteria. An HA tag was included immediately upstream of DWNN-GG for immunodetection using anti-HA antibodies; the construct was designed in such as way that it could be re-used to generate HA-tagged versions of existing constructs cloned into pGEX-6P-2. The above findings lay the foundation for future structural and functional studies of the involvement of RBBP6 in regulation of the cancer-related proteins p53 and YB-1, which may have far-reaching consequences in the fight against cancer. / National Research Foundation (NRF)