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Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. It has been established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesized that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. In support of the hypothesis we found that glucosamine-supplemented apoE-/- mice had elevated levels of ER stress and atherosclerosis. Mechanistically, our data showed that glucosamine induced ER stress by interfering with the lipid-linked oligosaccharide biosynthesis pathway and protein N-glycosylation. These findings support a model by which conditions of hyperglycemia promote vascular complications through a glucosamine-intermediate. / Thesis / Doctor of Philosophy (PhD) / Diabetes mellitus dramatically increases the risk for heart attacks and strokes. High blood glucose is utilized in cells through its conversion into metabolites, such as glucosamine. We hypothesized that conditions of high blood glucose can led to an increase in intracellular glucosamine which can initiate pathways involved in accelerating atherosclerosis. Our results show that this is possible in both human cells and mice.

Essential hypertension is the leading contributor to premature death worldwide. Endoplasmic reticulum (ER) stress has recently been implicated in diseased blood vessels and hypertension. It is unclear whether ER stress is a cause or a consequence of hypertension. We hypothesized that ER stress inhibition would prevent the development of hypertension in the young spontaneously hypertensive rat (SHR) by improving vascular structure and function. The SHR was used as a genetic model of human essential hypertension, and the Wistar Kyoto (WKY) rat as its normotensive control. The first study conducted involved assessing the levels of ER stress in young SHRs, before they developed hypertension. The second study conducted involved treating rats with 1g/kg/day of the sodium salt of 4-phenylbutyric acid (4-PBA) orally for 8 weeks from 5 weeks of age. Blood pressure was measured weekly, noninvasively via radiotelemetry. Mesenteric arteries were collected at sacrifice. Finally, the third study conducted involved treating rats with 1g/kg/day 4-PBA orally for eight weeks from five weeks of age, and then withdrawing the drug for four weeks to determine if drug treatment created a sustained lowering of blood pressure. In the first study, ER stress markers were observed to be significantly increased in the young SHR when compared to the WKY. In the second study, blood pressure was observed to be significantly lower in the 4-PBA-treated SHR groups than in the untreated SHRs. In addition, mesenteric arteries from the 4-PBA treated SHRs had a significant decrease in media/lumen ratio, ER stress marker expressions, as well as improved vasodilatory response to carbachol and reduced contractile responses to phenylephrine. In the third study, 4-PBA was able to keep the blood pressure low for one week after withdrawal, however, blood pressure returned to similar levels as untreated SHRs by the end of three weeks. Overall, ER stress inhibition, via 4-PBA, blunted the development of hypertension in the SHR. / Thesis / Master of Science (MSc)

Sss1p is an essential component, along with Sec61p, of the protein conducting channel (PCC) in the ER of the yeast Saccharomyces cerevisiae. It belongs to a family of proteins termed tail-anchor (TA) proteins. The TA consists of a single hydrophobic sequence at the carboxyl-terminus which anchors the protein to the membrane in a Type II (N_cytoplasm-C_lumen) orientation. TA proteins are targeted to their membranes of function through an uncharacterized SRP-independent, post-translational mechanism. The targeting mechanism and function of Sss1p are not known. In this thesis, results will be presented from targeting and functional studies of Sss1p. Sss1p is predicted to contain an ER targeting signal similar to mammalian VAMP-1A. Disruption of this putative signal caused incomplete mislocalization of Sss1p to the mitochondria which did not affect yeast growth. Mutations in the TA of Sss1p had numerous effects. Yeast expressing these mutants showed diminished growth, a defect in co-and post-translational translocation, inefficient ribosome binding to Sec61 p and the mislocalization of translocon components from light membranes (predominantly ER) to heavy membranes (predominantly mitochondria). It is argued that mutations in the TA of Sss1p disrupt the function of the protein, subsequently leading to the general defects listed above. Two possible functions for Sss1p are proposed: Sss1p is involved in forming the signal sequence binding pocket of the translocon and/or is essential for the integrity of the PCC. / Thesis / Master of Science (MS)

The proprotein convertase subtilisin/kexin type 9 (PCSK9) was first characterized in 2003 by Seidah and colleagues and marked the beginning of what is now considered by many as the greatest advancement in the field of cardiovascular disease (CVD) research since the discovery of the LDLR nearly half of a century ago. Since its discovery, PCSK9 was shown to enhance the degradation of cell-surface low-density lipoprotein (LDL) receptor (LDLR) and gain-of-function (GOF) mutations were shown to correlate with CVD risk. In contrast, patients carrying loss-of-function (LOF) mutations in PCSK9 highlighted a novel therapeutic approach for LDL lowering as they exhibit a life-long state of hypocholesterolemia and reduced CVD risk. A decade after the cloning of the PCSK9 gene, pharmaceutical companies have now developed a variety of PCSK9 inhibitors, ranging from monoclonal antibodies (mAbs) to small interfering RNA (siRNA) and vaccines, which have been shown to markedly reduce LDL cholesterol levels in pre-clinical models, as well as in patients at high risk of CVD. Despite these advances, there remained several unanswered questions regarding the mechanisms by which PCSK9 expression and secretion is regulated in the liver; the tissue from which the circulating pool of PCSK9 almost exclusively originates. The thought that further development of our understanding of PCSK9 biology may lead to the discovery of a signaling cascade that could be targeted by small molecules, the only class of inhibitor that has not yet been developed, has now merited additional research attention. The focal point of my doctoral studies represents the axis between a cellular process known as endoplasmic reticulum (ER) stress and PCSK9 expression/biosynthesis. ER stress is a deleterious cellular process that is known to occur in secretory cell types, such as liver hepatocytes, and is a well-established causative driver of an array of human diseases ranging from CVD to neurodegenerative diseases. ER stress is prevalent in the livers of patients with metabolic disease and is also known to activate the transcription factor capable of regulating PCSK9 levels, the sterol regulatory element-binding protein 2 (SREBP2). Based on this information, the first aim during the course of my PhD studies was to determine whether ER stress affected the expression and secretory status of PCSK9. In the past several years, I demonstrated that ER stress caused by ER Ca2+ depletion led to a marked increase in PCSK9 protein expression, but blocked its secretion as a result of its retention in the ER. Such a result was also associated with heightened hepatic LDLR expression and reduced LDL cholesterol levels in mice. Additional studies also characterized a variety of agents, including caffeine, as potent inhibitors of PCSK9 expression via increasing ER Ca2+ levels, which antagonized SREBP2 activity. As our initial studies revealed ER PCSK9 retention as a viable strategy for PCSK9 inhibition and LDL lowering, follow-up studies were also carried out to determine the outcome of such a strategy on liver function and injury. Given that heritable mutations in proteins that transit the ER can accumulate in this compartment and cause ER storage disease (ERSD), it was critical to further evaluate whether ER PCSK9 retention would lead to a similar outcome. In a series of experiments with rather surprising outcomes, we observed that the retention of the LOF Q152H PCSK9 mutant in the ER failed to cause ER stress; even in mice overexpressing the protein. Interestingly, tissue culture and mouse models demonstrated that the retention of PCSK9 in this cellular compartment increased the cellular abundance of ER stress response chaperones, such as the glucose-regulated proteins of 78- and 94-kDa (GRP78 and GRP94, respectively), but did not activate transducers of the ER stress signaling cascade. Strikingly, mice expressing the ER-retained PCSK9 Q152H mutant were protected against ER stress, suggesting a novel co-chaperone-like role of intracellular PCSK9. Collectively, the ER environment including secondary messengers like Ca2+ as well as its chaperones, plays a critical regulatory role on PCSK9 expression and secretion. Agents that increase ER Ca2+ levels can be utilized to block PCSK9 expression at the mRNA level to increase hepatic LDL clearance, and ER PCSK9 retention may also represent a safe avenue with a similar LDL lowering outcome. Beyond LDL lowering, hepatic ER PCSK9 retention may also serve as a novel strategy to enhance ER function and protect against ER stress-driven diseases of the liver. / Thesis / Doctor of Philosophy (Medical Science)

Objetivos: Este estudo teve como objetivo avaliar a resistência de união entre cimento resinoso e as amostras de zircônia irradiadas como o laser de Er,Cr:YSGG em quatro parâmetros. Foram utilizadas 78 unidades experimentais (12,4 mm x 7,6 mm x 1,6 mm) a partir de blocos de Y-TZP (zircônia tetragonal estabilizada com ítrio) pré-sinterizados. A fim de obter superfícies padronizadas, os espécimes foram lixados com uma sequência decrescente de lixas (600, 1200, 1500). Em seguida, os blocos foram sinterizados em um forno específico, de acordo com as recomendações do fabricante. Os grupos experimentais foram compostos a partir de um fator de variação - tratamento de superfície. Foram formados 6 grupos experimentais (n=13) Controle, sem tratamento de superfície, grupo Rocatec, com tratamento triboquímico (Rocatec System Plus 3M ESPE), e 4 grupos laser (Waterlase Iplus Biolase Inc. Irvine, CA, EUA comprimento de onda 2780nm, 700?s, de 50% de ar e 50% de água, ponta MZ6 600?m): L3W 20Hz (53,57J/cm2), grupo L3W 50Hz (21,43J/cm2), grupo L6W 20Hz (107,14J/cm2), grupo L6W 50Hz (42,68J/cm2). Um espécime de cada grupo foi analisado através de microscopia eletrônica de varredura MEV (10000x). Após dos tratamentos de superfície, em cada bloco de zircônia foram colados 6 cilindros de resina usando cimento dual contendo MDP (Panavia F 2.0). Todas as amostras foram armazenadas em água destilada a 37ºC. O teste de microcisalhamento foi feito na máquina de ensaio universal (Instron 5565 EUA). Três dos 6 tubos colados em cada bloco foram avaliados depois de 24 horas de armazenamento em água a 37ºC e os 3 restantes foram avaliados depois de 2000 ciclos térmicos (5ºC - 30ºC). O tipo de falha foi determinado através de estereomicroscópio. Os dados foram analisados pelos testes de análise de variância (ANOVA) e de Tukey com um nivel de significância de p<0,01. Resultados: O grupo controle positivo (ROCATEC) apresentou diferença significativa (p <0,001) com os demais grupos. Quando foi simulado o envelhecimento térmico (termociclagem), todos os grupos apresentaram diferença significativa (p <0,001) na resistência de união comparada com o protocolo sem termociclagem. O tratamento de superfície convencional ainda é preconizado como uma alternativa válida para melhorar a resistência de união na zircônia. O uso de laser de Er,Cr:YSGG com uma largura de pulso de 700?s dentro dos parâmetros estudados não promoveu mudanças na superfície de zircônia que favorecessem a adesão com cimento resinoso. A termociclagem influenciou negativamente em todos os grupos experimentais. / This study aimed to evaluate the bond strength between resin cement and the irradiated zirconia samples a the Er, Cr: YSGG laser in four parameters. It was used 78 experimental units (12.4mm x 7.6mm x 1.6mm) from pre-sintered Y-TZP (Ytriumstabilized Tetragonal Zirconia) blocks. In order to obtain standardized surfaces the specimens were sanded with a decreasing sanding sequence (600,1200,1500). Then, the blocks form sintered in a specific oven, according to the manufacturer\'s recommendations. The experimental groups were composed of a variation factor - the surface treatment. Thus, 6 experimental groups were formed (n=13). Control, without surface treatment, the Rocatec group, with triboquimic treatment (Rocatec System Plus 3M ESPE), and 4 laser groups (Waterlase Iplus Biolase Inc. Irvine, CA USA wavelength 2780nm, 700?s, 50% air and 50% water, MZ6 600?m tip): L3W 20Hz (53.57J / cm2), L3W 50Hz group (21.43J / cm2), L6W 20Hz group (107.14J / cm2), group L6W 50Hz (42.68J / cm2). One specimen from each group was analyzed by Scanning Electron Microscopy (10000x). After the surface treatments, 6 resin cylinders were bonded to each zirconia block using dual cement containing MDP (Panavia F 2.0). All samples were stored in distilled water at 37 ° C. The micro-shear test was performed on the universal test machine (Instron 5565 USA). 3 of the 6 tubes pasted in each block were evaluated after 24 hours of storage in 37 ° C water and the remaining 3 were evaluated after 2000 thermal cycles (5 ° C - 30 ° C). The type of failure was determined by stereomicroscope. The data were analyzed by a test for analysis of variance (ANOVA) and tukey with a significance level of p <0.01. Results: The positive control group (ROCATEC), evaluated immediately after cementation, showed a better bond strength and there was no significant difference when thermal aging was simulated (thermocycling), nor did it differ significantly from the groups without thermocycling of 3w20Hz or 6w50hz. Conventional surface treatment is still advocated as a valid alternative to improve bond strength in zirconia. The use of Er, Cr: YSGG laser with a pulse width of 700?s within the studied parameters did not promote changes in the zirconia surface that favored adhesion with resin cement. Thermocycling influenced negatively all experimental groups.

O crescente desenvolvimento e aprimoramento das técnicas e materiais odontológicos introduziu a tecnologia laser nas diversas especialidades da Odontologia. Ainda que existam poucos estudos na literatura quanto ao emprego do laser de Er,Cr:YSGG para o tratamento preventivo de lesões de cárie, acredita-se que a sua aplicação sobre a estrutura dental seja capaz de promover uma superfície mais resistente a desafios ácidos. O objetivo deste estudo foi avaliar o efeito da irradiação do esmalte dental humano com o laser de Er,Cr:YSGG, com diferentes densidades de energia, em relação à desmineralização. Quarenta e cinco blocos de esmalte dental de terceiros molares humanos (3 x 3 mm) foram aleatoriamente divididas em 5 grupos (n=9): G1 – Laser de Er,Cr:YSGG 0,25 W, 20 Hz, 2,84 J/cm 2 , G2 – – Laser de Er,Cr:YSGG 0,50 W, 20 Hz, 5,68 J/cm 2 , G3 – Laser de Er,Cr:YSGG 0,75 W, 20 Hz, 8,52 J/cm 2 , G4 – Tratamento com dentifrício fluoretado (controle positivo), G5 – Sem tratamento (controle negativo). Em seguida, as amostras&#3338;foram submetidas a ciclagem de pH por 2 semanas, permanecendo, diariamente, 6 h e 18 h nas soluções desmineralizante e remineralizante, respectivamente. Após o desafio ácido, as amostras foram seccionadas e o teste de Microdureza Knoop (25 g, 30 seg) foi realizado em distancias pré-determinadas em relação a superfície do esmalte (15 µm - 300 µm). OS valores de ANOVA e teste de Student Newman Keuls foram realizados(&#945; =5%). Os percentuais de inibição de cárie encontrados foram de: G1- 36,64 %, G2-37,98 %. G3 – 63,83% e G4 – 50,47 %. Não houve diferenças estatisticamente significativas entre a porcentagem de volume mineral perdido nos grupos G1 (1391,81±521,65) e G2 (1291,90±656,49), porém ambos foram maiores do que o grupo G3 (753,32±287,07). Nenhum dos grupos submetidos ao tratamento com o laser de Er,Cr:YSGG diferiu estatisticamente do grupo controle positivo (G4). Os resultados deste estudo in vitro sugerem que a irradiação com o Laser de Er,Cr:YSGG com densidade de energia de 8,52 J/cm 2 pode ser uma alternativa efetiva na obtenção do aumento da resistência ácida do esmalte e que densidades de energia menores podem apresentar um potencial cariostático semelhante ao tratamento com dentifrício fluoretado. / The continuing development and improvement of the techniques and materials have introduced the laser technology in the different specialties of Dentistry. Even though there are still few studies on the use of the Er,Cr:YSGG Laser on caries prevention, one believes that the enamel irradiation with this wavelength can lead to a more acid resistant surface. The aim of the present study was to evaluate the effect of different parameters of the Er,Cr:YSGG Laser irradiation on the enamel mineral loss. Forty five enamel samples obtained from third molars (3 x 3 mm) were randomly divided in 5 groups (n=9): G1 – Er,Cr:YSGG Laser 0.25 W, 20 Hz, 2.84 J/cm 2 , G2 – Er,Cr:YSGG Laser 0.50 W, 20 Hz, 5.68 J/cm 2 , G3 – Er,Cr:YSGG Laser 0.75 W, 20 Hz, 8.52 J/cm 2 , G4 – Treatment with fluoride toothpaste (positive control), G5 – No treatment (negative control). After the surface treatment, the samples were submitted to a 2-weeks pH-cycling, consisted of the daily immersion in demineralizing and remineralizing solutions for 6 h e 18 h, respectively. After&#3338;the acid challenge, the samples were sectioned and the Knoop microhardness test was performed (25 g, 30 sec) at different depth from the enamel surface (15 µm - 300 µm). ANOVA and Student Newman Keuls tests were performed (&#945; =5%). The percentage of lesion inhibition for each group was: G1- 36.64 %, G2- 37.98 %. G3 – 63.83% e G4 – 50.47 %. Regarding the percentage of mineral loss volume, Groups G1 (1391.81±521.65) and G2 (1291.90±656.49) did not differ from each other, but both were higher than group G3 753.32±287.07). All the groups irradiated with the Er,Cr:YSGG were similar to the positive control group (G4). The findings of the present study suggest that the Er,Cr:YSGG laser at 8.52 J/cm 2 can be an alternative for the enhancement of enamel acid resistant and that lower energy densities seem to present a cariostatic potential comparable to the use of fluoride dentifrice.

A irradiação com o laser de Er,Cr:YSGG promove aumento da área de superfície do esmalte dental irradiado, o que pode resultar em uma maior retenção e um efeito prolongado do fluoreto (F-) presente em produtos fluoretados de diferentes concentrações. O produto formado na superfície de esmalte originado de uma única aplicação de flúor fosfato acidulado (FFA 12.300 &mu;g F-/g) ou da frequente aplicação tópica de dentifrício contendo 1.100 &mu;g F-/g poderia ter seu efeito cariostático prolongado, pelo aumento de sua retenção na superfície do esmalte dental irradiado. Uma vez que o esmalte dentário livre de biofilme não sofre desmineralização na cavidade bucal, sugerimos um estudo in situ onde se possa avaliar o prolongamento do efeito do destas associações, também na presença de placa. As condições de irradiação do estudo in situ, foram determinadas, in vitro, com laser Er,Cr:YSGG no esmalte de maneira isolada ou combinada com as aplicações tópicas de: 1- dentifrício de concentração 1.100 &mu;g F-/g ou 2- FFA, para posteriores análises da formação e retenção de CaF2. Foram realizadas análises morfológicas por microscopia eletrônica de varredura, determinação da concentração do flúor solúvel em álcali por meio do eletrodo íon específico e análise da microdureza em corte longitudinal. Os resultados por microscopia eletrônica de varredura verificaram qualitativamente a formação de produtos na superfície de esmalte na forma de CaF2. A análise bioquímica para determinação quantitativa do F- solúvel em álcali determinou como sendo estatisticamente diferentes (p&le;0,05) os Grupos nos quais o laser foi utilizado previamente à aplicação tópica dos dois tipos de produtos fluoretados de diferentes concentrações (dentifrício e FFA), in vitro. Em seguida, foi realizado o estudo in situ quando voluntários utilizaram dispositivos palatinos, contendo blocos de esmalte humano, previamente tratados, com o objetivo de acúmulo da placa nativa sobre os mesmos. Durante a fase in situ, os voluntários permaneceram utilizando dentifrício F- para verificação da ação do mesmo na presença de biofilme sobre os blocos irradiados. Foram correlacionados os efeitos da formação de F-, decorrentes dos tratamentos propostos, na redução da desmineralização. A análise bioquímica para quantificação do F- solúvel em álcali determinou como sendo estatisticamente diferentes (p0,05) os Grupos nos quais o laser foi utilizado após a aplicação tópica dos dois tipos de produtos fluoretados de diferentes concentrações (dentifrício e FFA), in situ, sugerindo um efeito prolongado da sinergia dos tratamentos na diminuição da desmineralização. / The effect of the Er, Cr: YSGG laser promotes increased surface enamel area, which can result in increased retention and prolonged effects of Fluoride (F-) present in products with different concentrations of fluoride. The cariostatic effect from product formed in the enamel surface originated from a single application of acidulated phosphate fluoride (APF 12 300 &mu;g F-/ g), or frequent topical application of dentifrice containing 1,100 &mu;g F-/ g, could be prolonged by increasing its retention on irradiated enamel surface. Once the biofilm-free enamel does not suffer demineralization within the oral cavity, it is proposed an in situ study where we can evaluate the prolongation of the effect of these associations, also in the presence of plaque. The irradiation conditions of the in situ study were determined in vitro with Er, Cr: YSGG laser irradiation of enamel surface either alone or combined with one of the topical applications: 1 - dentifrice F-1,100 &mu;g / g or 2 - APF For further analysis of the formation and retention of CaF2. Morphological analyzes were performed by scanning electron microscopy, determination of the concentration of alkali-soluble fluoride by specific ion electrode analysis and microhardness. The results of scanning electron microscopy verified qualitatively the formation of products in the enamel surface in the form of CaF2. Biochemical analysis for quantitative determination of F-soluble in alkali determined to be statistically different (p&le;0.05) Groups in which the laser was used prior to application of topical fluoride products of two types of different concentrations (APF and dentifrice) in vitro. Then, the study was conducted in situ when volunteers wore palatal appliances containing blocks of human enamel, pretreated aiming native plaque formation. During in situ experiment, the volunteers remained using F-dentifrice. Correlations with the effects of F-formation, resulting from treatments proposed in the reduction of demineralization were made. Biochemical analysis for quantitative determination of F- alkali soluble determined to be statistically different (p 0.05). Groups in which the laser was used after topical application of both types of different fluoride concentrations (APF and toothpaste), in situ, suggested an synergic effect, extending treatment efficiency in reducing demineralization.

O laser de Er,Cr:YSGG se apresenta como um aliado aos procedimentos minimamente invasivos. Porém, pouco se sabe sobre a longevidade da resistência de união à dentina irradiada por este equipamento. Dessa forma, o objetivo deste estudo foi avaliar a resistência de união à dentina irradiada após 90 dias de armazenamento. Para tanto, foram utilizados 64 molares humanos hígidos, que tiveram as superfícies oclusais desgastadas até a exposição de uma superfície de dentina. Esta superfície foi tratada com: abrasão com lixa SiC 600# (controle) e irradiação com o laser de Er,Cr:YSGG em 2W (90,9 J/cm2) ou 4W (181,8 J/cm2). Foram utilizados os seguintes sistemas adesivos: Adper Single Bond (3MESPE), Clearfil SE (Kuraray) ou Clearfil S3 (Kuraray), e a confecção de um bloco de resina composta Z250-3MESPE. Os dentes foram seccionados para a obtenção de palitos para o teste de microtração. Metade dos palitos foi ensaiada imediatamente, e a outra metade após 90 dias de armazenamento em água destilada. Os resultados mostraram que em todos os grupos em que foi utilizado o laser Er,Cr:YSGG apresentaram os menores valores de resistência adesiva. Com exceção do grupo controle/Clearfil S3, todos os grupos apresentaram decréscimo nos valores de resistência adesiva após os 90 dias de armazenamento. Entre os sistemas adesivos utilizados, o Single Bond apresentou os melhores valores de resistência adesiva, demonstrando maior estabilidade frente aos 90 dias de desafio/armazenamento. / In the minimally invasive dentistry, the Er,Cr:YSGG laser has been considered as an efficient equipment. However, little is known about the longevity of the bond strength to irradiated dentin. So, the objective of this study was to evaluate the bond strength values to laser irradiated dentin after 90 days storage. Sixty-four sound human molars were used. A flat coronal dentin surface was obtained by removing the oclusal enamel with a slow speed diamond saw. Dentin surfaces were treated with: abrasion with SiC 600# (control group) and laser irradiation with Er,Cr:YSGG in 2W or 4W. Subsequently, adhesive systems were used: Adper Single Bond (3MESPE), Clearfil SE (Kuraray) or ClearfilS3 (Kuraray). After adhesive procedures, a block of Z250-3MESPE composite resin was build up on each tooth. The teeth were sectioned in order to obtain samples for the microtensile bond strength test. Half of the samples was tested immediately, and the other half were stored for 90 days in distilled water. The results showed that all the groups in which laser was used presenter lower bond strength values. With the exception of Clearfil S3/control group, all groups showed weaker bond strength after 90 days of storage. Among all the adhesives systems used, Single Bond presented the best results, showing more stability after 90 days of storage.

No tratamento endodôntico, várias fases de igual importância são necessárias para a realização de uma terapêutica bem sucedida, entre elas podemos citar a instrumentação. Apesar de todas as técnicas e instrumentos propostos para a Endodontia, o sucesso esperado ainda não foi alcançado. Tanto a instrumentação manual quanto à rotatória não resulta na limpeza das áreas polares dos canais radiculares com achatamento mésio-distal. No intuito de obter melhor resultado clínico, avaliou-se “in vitro” a remoção da camada de “smear” das paredes dos canais radiculares de 40 dentes incisivos inferiores portadores de achatamento mésio-distal submetidos à instrumentação rotatória por meio da técnica “Free Tip Preparation”, utilizando o laser de Er: YAG nos parâmetros de 140 e 250 mJ. Para tanto, a fibra óptica do aparelho foi deslocada lentamente, com velocidade de 2 mm/s, até o orifício cervical na câmara pulpar, na parede vestibular e lingual da raiz; e sua eficácia na remoção da “smear” foi comparada aos grupos onde se utilizou apenas Hipoclorito a 2,5% e Hipoclorito de sódio a 2,5% alternado com EDTA a 17%. Os dentes foram analisados por meio de microscopia eletrônica de varredura, e as fotomicrografias das áreas mais representativas dos terços médio e apical foram avaliadas por três examinadores diferentes, com grau mínimo de Mestre, que observaram a quantidade da camada de “smear” presente nas amostras em relação a três padrões. Os examinadores, sem que tivessem o conhecimento prévio de qual dos grupos estavam avaliando, atribuíram escores de 1 a 4 as fotomicrografias, de acordo com a quantidade da camada de “smear” visualizada. Os dados obtidos foram submetidos à análise estatística não-paramétrica, comparando-se os diferentes grupos estudados (teste de Kruskal-Wallis) e os terços radiculares (teste de Wilcoxcon). Os resultados mostraram que, quanto à quantidade da “smear” encontrada nos grupos estudados, o grupo que utilizou Hipoclorito a 2,5% alternado com EDTA apresentou os canais radiculares mais limpos seguido do grupo do laser 250 mJ e posteriormente pelos grupos do laser Er: YAG a 140 mJ e do hipoclorito a 2,5%, que não apresentaram diferença estatisticamente significante entre si. Quanto aos terços, o apical apresentou maior quantidade da “smear” em comparação com o terço médio (p< 0,01). / Manual or rotary instrumentation techniques do not efficiently clean mesio-distal flattened root canals. This study evaluated, in vitro, smear layer removal of 40 mesio-distal flattened root canals after rotary instrumentation. The teeth were then divided into 4 groups: group 1: rotary instrumentation and 2.5% sodium hypochlorite used as irrigating solution; group 2: instrumented as group 1 and followed by Er: YAG laser irradiation (140mJ input/10Hz), withdrawn at 2mm/s from the apical to the cervical region touching the buccal walls the procedure was then repeated for the lingual wall); group 3: instrumented as group 1 and followed by Er: YAG laser irradiation (250mJinput/10Hz) withdrawn at 2mm/s from the apical to the cervical region touching the buccal walls the procedure was then repeated for the lingual wall) and group 4: instrumented as group 1 and alternated with 17% EDTA Teeth were split longitudinally and prepared for examination under scanning electron microscopy. Scores from 1 to 4 were given to the fotomicrographs by three independent evaluators, and these data was submitted to statistical analysis. The teeth where alternated 17% EDTA were used (group 4) showed less amount of smear layer, followed by the group irradiated with 250mJinput/10Hz (group 3), the group irradiated with 140mJ input/10Hz and the group where only sodium hypochlorite was used. The apical third presented more smear layer than the middle third (p<00.1).