O controle da extrusão apical durante a reintervenção endodôntica é essencial para o sucesso do novo tratamento. Nesse contexto, o presente estudo teve como objetivo comparar a quantidade de material sólido extruído na desobturação de canais radiculares variando-se a técnica de esvaziamento e o limite de obturação. Foram utilizados 40 incisivos inferiores previamente tratados divididos em dois grupos de acordo com o limite de obturação estabelecido. Cada grupo foi subdividido em dois subgrupos levando-se em conta a técnica de desobturação empregada; manual (subgrupos A1 e B1) e mecânico-rotatória com limas de Ni-Ti (Quantec LX) (subgrupos A2 e B2). O material sólido extruído foi coletado por meio do sistema de filtração Millipore, levado à secagem em dessecador de sílica e pesado em balança analítica de precisão. Os resultados obtidos foram submetidos a ANOVA para dois fatores de variação sendo em seguida empregado o Teste de Tukey (&#945; = 5%). A técnica de desobturação mecânico-rotatória produziu menor extrusão (0,66mg) que a manual (1,11mg), havendo diferença estatística significante entre elas (p < 0,05). Os canais preenchidos até o vértice radiográfico apresentaram maior quantidade de extrusão (1,38mg) do que os obturados 1 mm aquém do forame (0,39mg), observando-se diferença estatística significante entre eles (p < 0,05). A menor quantidade extrusão foi observada no subgrupo A2 (0,20mg), em que foi empregada a técnica rotatória de desobturação em canais obturados 1mm aquém do forame apical, sendo constatada diferença estatisticamente significante deste subgrupo com os demais (p < 0,05). A extrusão de material sólido durante a desobturação de canais radiculares é influenciado pela técnica empregada e pelo limite apical de obturação. / The apical extrusion control during the endodontic retreatment is essential for the success of the new treatment. The purpose of this study was to compare the quantity of solid apically extruded material during filling removal according the gutta-percha removal technique and root filling limit. Forty mandibular incisors with a single straight canal were selected. The canals were previously endodontically treated and then divided into two groups according the filling level. Each group was subdivided in two groups considering the retreatment technique: stainless steel hand files (subgroups A1 and B1) versus niquel-titanium rotatory instruments (subgroups A2 and B2). The extruded solid material was collected by Millipore filtration system, dried in silica desiccators and weighed in an eletrobalance. The results were analyzed using ANOVA with two variation factors and Tukey Test (&#945; = 5%). The niquel-titanium rotatory instruments produced less extrusion (0,66mg) than the stainless steel hand files (1,11mg), with significant statistical difference between them (p < 0,05). The canals filled until the radiographic apex showed larger amount of extruded material (1,38mg) than those filled 1 mm beyond the foramen (0,39mg). It was observed significant statistical difference between them (p < 0,05). The smaller extruded debris amount was observed in subgroup A2 (0,20mg), in which one the rotary technique was used to remove the gutta-percha of canals filled 1mm beyond the apical foramen. It was verified significant statistical difference of this subgroup with the other ones (p < 0,05). The extrusion of solid material during the gutta-percha removal is influenced by the technique as well as the apical filling limit.

Considerando-se a localização intra-óssea das lesões periapicais, o diagnostico clinico e dificultado e a avaliação radiográfica não fornece informações suficientes para o diagnostico de um periodonto apical sadio pós-tratamento. Dessa maneira, o objetivo deste estudo foi comparar os achados radiográficos e por tomografia computadorizada de feixe cônico com a avaliação microscópica após tratamento de canais radiculares em dentes de cães e avaliar a participação das metaloproteinases da matriz (MMPs) na lesão periapical e nos tecidos em processo de reparação, assim como em cistos e granulomas periapicais obtidos de humanos. A seguir, os mecanismos envolvidos na cementogênese apical foram investigados utilizando células do ligamento periodontal de humanos. Foram induzidas lesões periapicais em dentes de cães e o tratamento endodôntico foi realizado em sessão única ou apos utilização de um curativo de demora a base de hidróxido de cálcio [Ca(OH)2]. Avaliações radiográficas e tomográficas foram realizadas previamente, após a indução das lesões periapicais e 180 dias apos o tratamento endodôntico. Os tecidos periapicais foram avaliados por meio de microscopia de luz, imunofluorescência, imunoistoquímica e RT-PCR em tempo real. In vitro, células do ligamento periodontal foram utilizadas para avaliar os efeitos da estimulação com Ca(OH)2 nos processos de migração, proliferação, diferenciação celular e mineralização. As vias de sinalização envolvidas na diferenciação cementoblastica foram investigadas por meio de inibidores bioquímicos da via das proteínas quinases ativadoras de mitose (MAPK), bloqueadores de canais de cálcio e silenciadores de RNA para proteínas quinases reguladas por sinal extracelular (ERK-1 / ERK-2). De acordo com os resultados obtidos, a tomografia computadorizada permitiu a detecção de lesões periapicais com maior sensibilidade e acuracia que a radiografia periapical convencional, utilizando-se a avaliação microscópica como padrão-ouro. Histologicamente, as lesões periapicais experimentalmente induzidas apresentaram bactérias distribuídas pelo sistema de canais radiculares e lacunas de reabsorção do cemento e estavam associadas a desorganização das fibras colágenas e alta expressão de MMPs. A presença das MMPs em processos inflamatórios periapicais de humanos (cistos e granulomas) foi confirmada. Nos dentes com lesão periapical submetidos ao tratamento endodôntico em sessão única o desfecho do tratamento endodôntico foi caracterizado pela manutenção ou progressão da lesão periapical e alta expressão de MMPs. Por outro lado, nos dentes submetidos ao tratamento endodôntico utilizando o Ca(OH)2 como curativo de demora, maior numero de espécimes apresentaram regressão da lesão periapical e houve modulação da expressão de MMPs pelo tratamento. Neste grupo foi evidenciada neoformação de cemento no forame apical. Células do ligamento periodontal estimuladas com Ca(OH)2 expressaram proteínas especificas de cementoblastos (CEMP-1, CAP) e foram capazes de sintetizar nódulos de mineralização, mediados via ERK MAPK. A ação do Ca(OH)2 ocorreu via canais de cálcio, uma vez que o bloqueio destes canais inibiu a fosforização de ERK-1 e ERK-2 e, portanto, a expressão de CEMP-1 e CAP. CEMP-1 estimulou a migração, proliferação e mineralização mediada por células, exercendo um papel central na cementogenese, uma vez que o bloqueio de CEMP-1 inibiu a migração celular e mineralização. Juntos, estes resultados permitem concluir que a tomografia computadorizada e superior a radiografia periapical convencional para detecção de lesões periapicais refratarias ao tratamento de canais radiculares. Ainda, o tratamento endodôntico realizado utilizando o hidróxido de cálcio como curativo de demora propiciou um reparo apical e periapical mais favorável do que o tratamento endodôntico em sessão única, possivelmente devido a capacidade do Ca(OH)2 induzir a diferenciação de células do ligamento em células com um fenótipo cementoblastico e posterior mineralização. / Clinical diagnosis of apical periodontitis is difficult due to the intraosseous nature of the disease and radiographic evaluation does not provide sufficient information to determine a healthy apical periodontium following root canal therapy. Therefore, the aim of this study was to compare the radiographic and cone beam computed tomographic findings with microscopic evaluation following root canal treatment in dogs teeth. Then, the presence of matrix metalloproteinases (MMPs) in apical periodontitis and during the healing phase following treatment was evaluated and compared to the expression of MMPs in periapical cysts and granulomas obtained from human. Finally, the mechanisms involved in apical cementogenesis were investigated using human periodontal ligament cells. Apical periodontitis was induced in dogs teeth and then root canal treatment was performed in a single visit or using calcium hydroxide [Ca(OH)2] as the root canal dressing. Tomographic and radiographic evaluations were performed prior to and following induction of apical periodontitis, and 180 days following root canal therapy. Periapical tissues were evaluated by conventional light microscopy, immunofluorescence, immunohistochemistry, and real time RT-PCR. In vitro, periodontal ligament cells were used to evaluate the effects of Ca(OH)2 treatment on cell migration, proliferation, differentiation, and mineralization. The signaling pathways triggered by treatment with Ca(OH)2 were investigated using mitogen activated protein kinase (MAPK) biochemical inhibitors, calcium channel blockers, and extracellular regulated protein kinase (ERK-1 / ERK-2) silencing RNAs. Based on the results obtained, apical periodontitis was detected with higher sensitivity and accuracy by means of cone beam computed tomography compared to conventional periapical radiographs, using microscopic evaluation as the gold standard. Histological evaluation revealed that teeth with apical periodontitis presented microorganisms throughout the root canal system and in areas with resorption of cementum. Apical periodontitis was characterized by collagen fiber disorganization and high expression of MMPs. The presence and activity of MMPs was confirmed in periapical inflammatory diseases (cysts and granulomas) obtained from humans. Root canal treatment outcome was characterized by maintenance or progression of apical periodontitis and high expression of MMPs in teeth submitted to root canal treatment in a single visit, whereas root canal treatment outcome in teeth submitted to root canal treatment using Ca(OH)2 as the root canal dressing, higher number of teeth presented reduced apical periodontitis and lower expression of MMPs. In this group, cementum neogenesis was evident in the apical foramina. Periodontal ligament cells stimulated with Ca(OH)2 expressed cementoblastic specific proteins (CEMP-1, CAP) and were able to synthesize mineralized nodules via ERK MAPK. The effects of Ca(OH)2 occurred via calcium channels, since their blockade prevented ERK-1 and ERK-2 phosphorylation and therefore expression of CEMP-1 and CAP. CEMP-1 stimulated cell migration, proliferation, and mineralization and it was central to cementogenesis because blockade of activity of CEMP-1 prevented cell migration and mineralization. Taken together, these findings demonstrate that cone beam computed tomography is superior to conventional periapical radiography for detection of refractory apical periodontitis. Furthermore, the root canal treatment using Ca(OH)2 as the root canal dressing permitted a more favorable outcome than the root canal treatment performed in a single visit, probably due to the ability of Ca(OH)2 induce cementoblastic differentiation of periodontal ligament cells and mineralization.

Afin de résoudre la problématique des substituts osseux faiblement vascularisés, un des challenges majeurs en ingénierie tissulaire osseuse est de favoriser le développement précoce d’une microvascularisation. La reproduction du microenvironnement local et l’organisation cellulaire in situ sont des approches innovantes pour optimiser la formation osseuse. En Biofabrication, la Bioimpression Assistée par Laser (LAB) est une technologie émergente permettant l’impression de cellules et de biomatériaux avec une résolution micrométrique. L’objectif de ce travail était d’étudier l’effet de l’organisation de la pré-vascularisation par LAB sur la régénération osseuse. La station de bioimpression Novalase a été utilisée pour imprimer des motifs de cellules endothéliales sur un « biopaper » constitué de collagène et de cellules souches issues de la papille apicale. Les paramètres d’impression, densités cellulaires et conditions de recouvrement ont été optimisés afin de favoriser la formation d’un réseau microvasculaire avec une architecture définie in vitro. Ce modèle a ensuite été transposé in vivo, grâce à la bioimpression in situ de cellules endothéliales au niveau de défauts osseux critiques chez la souris, afin d’évaluer si la prévascularisation organisée par LAB permettait de promouvoir et contrôler spatialement le processus de régénération osseuse. Les résultats ont montré que la bioimpression permettait d’augmenter la densité de vaisseaux dans les défauts osseux et de favoriser la régénération osseuse. / In order to solve the issue of poorly vascularized bone substitutes, development of a microvasculature into tissue-engineered bone substitutes represents a current challenge. The reproduction of local microenvironment and in situ organization of cells are innovating approaches to optimize bone formation. In Biofabrication, Laser-Assisted Bioprinting (LAB) has emerged as a relevant method to print living cells and biomaterials with micrometric resolution. The aim of this work was to study the effect of prevascularization organized by LAB on bone regeneration. The laser workstation Novalase was used to print patterns of endothelial cells onto a « biopaper » of collagen hydrogel seeded with stem cells from the apical papilla. Printing parameters, cell densities and overlay conditions were optimized to enhance the formation of microvascular networks with a defined architecture in vitro. This model was then transposed in vivo, through in situ bioprinting of endothelial cells into mouse calvarial bone defects of critical size, to investigate if prevascularization organized by LAB can promote and spatially control bone regeneration. The results showed that bioprinting allowed to increase blood vessel density in bone defects and promote bone regeneration.

A citricultura se destaca como uma das mais importantes atividades do agronegócio brasileiro, sendo o estado de São Paulo o principal produtor mundial de laranja e o maior exportador de suco concentrado e congelado. O psilídeo dos citros (Diaphorina citri) tornou-se nos últimos anos um dos mais importantes vetores na cultura, devido à transmissão de \'Candidatus Liberibacter asiaticus\' e \'Ca. L. americanus\', bactérias associadas ao huanglongbing (HLB), principal doença da citricultura atual. Uma vez que não há variedades comerciais de citros resistentes ao HLB, seu manejo é baseado no uso de mudas sadias, erradicação de plantas infectadas e pelo controle químico do vetor. No entanto, o custo elevado e os significativos danos ambientais causados pelos inseticidas químicos, associados à possibilidade da seleção de populações de psilídeos resistentes a esses produtos, têm levado à busca por estratégias alternativas de manejo do vetor do HLB que sejam mais adequadas e sustentáveis. Nosso grupo tem tentado contribuir nesse sentido, com a prospecção e caracterização de estirpes de Bacillus thuringiensis (Bt) patogênicos ao psilídeo. Sendo assim, os objetivos deste estudo foram: (a) Confirmar a capacidade endofítica e a patogenicidade de estirpes de Bt em seedlings e mudas de citros com diferentes combinações de copa/porta-enxerto; (b) Determinar a concentração letal (CL50) necessária para ocasionar mortalidade em populações de D. citri; (c) Estudar a interação entre toxinas Cry e receptores de membrana do intestino de ninfas e (d) Avaliar a compatibilidade das estirpes selecionadas com agrotóxicos comumente utilizados na citricultura. Os bioensaios realizados com seedlings e mudas de citros demonstraram que as estirpes de Bt (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 e S1989) translocaram endofiticamente nas plantas, mantendo sua viabilidade e, em sua maioria, o potencial patogênico para ninfas de D. citri. Para seedlings, os valores de mortalidade passaram de 80% para as estirpes S1302 e S1450. Foram observados esporos e células vegetativas de B. thuringiensis subsp. kurstaki expressando green fluorescent protein (Btk::GFP), visualizados por microscopia de fluorescência, aderidos principalmente aos elementos de vasos e no xilema, obtidas de amostras seccionadas transversal e longitudinalmente, de caule e raiz de seedlings e mudas de laranjeira Pera. A CL50 e CL80 para a estirpe S1302 foi de 4,92 × 104 esporos/mL e 6,63 × 107 esporos/mL, respectivamente. Já para a estirpe S1450, 50% de mortalidade das ninfas testadas foi estimada em 2,19 × 104 esporos/mL, e a CL80 foi de 6,18 × 108 esporos/mL, quando utilizadas suspensões de esporos da bactéria diretamente no substrato de seedlings de laranjeira Pera. Os ensaios de ligação demonstraram que todas as toxinas Cry presentes nas estirpes S1302, S1450 e S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B e Cry11) interagiram com os receptores de membrana intestinal, brush border membrane vesicles (BBMV\'s), obtidos de ninfas de D. citri. Ensaios in vivo evidenciaram que as estirpes S1302 e S1450 mostraram-se compatíveis com todos os agrotóxicos comumente utilizados na citricultura, ainda que os produtos à base de cobre e o inseticida Dimetoato tenham sido deletérios em aplicações diretas na bactéria in vitro. Esses dados evidenciam a possibildade do uso de Bt como bioinseticida no manejo integrado do HLB. / Citriculture is one the most important activities of Brazilian agribusiness, and the State of São Paulo being the world\'s leading orange producer and the largest juice exporter. Asian citrus psyllid - ACP (Diaphorina citri) has become one of the most important vectors in the citriculture in recent years, due to the transmission of \'Candidatus Liberibacter asiaticus\' and \'Ca. L. americanus\', bacteria associated with huanglongbing (HLB), the main citrus disease worldwide. Nowadays there are no commercial citrus varieties resistant to HLB, and its management is based on the use of healthy nursery citrus trees, eradication of symptomatic planst from the orchards, and vector chemical control. However, excessive cost and environmental damage due to application of chemical insecticides associated with the possibility of selection of ACP resistant populations, have led researchers to persue alternative strategies for the management of HLB. Our group has contributed with the screening of Bacillus thuringiensis (Bt) strains with potential against ACP. Therefore, our objectives were: (a) to confirm Bt endophytic translocation in citrus seedlings and nursery trees with different scion-rootstock combinations and to evaluate their pathogenicity against D. citri; (b) to estimate the lethal concentration (LC50 and LC80) to D. citri nymphs; (c) to study the interaction between Cry toxins and brush border membrane vesicle (BBMV) of the midgut D. citri nymphs, and (d) to evaluate the compatibility of pesticides with the selected Bt strains. The bioassays with citrus seedlings and nursery trees demonstrated that the Bt strains (Cyt1A, Cry2Aa, Cry4A, Cry10, Cry11, S1302, S1450 and S1989) translocated from roots to young leaves, maintaining their viability and pathogenicity against D. citri nymphs. For the seedlings, we found mortality values up to 80% for strains S1302 and S1450. Btk::GFP spores and vegetative cells were visualized by fluorescence microscopy in citrus seedlings and nursery trees adhered mainly to vessels and xylem, from roots to stems, in cross-section analyses. LC50 and LC80 for strain S1302 were 4.92 × 104 spores/mL and 6.63 × 107 spores/mL, respectively. For strain S1450, 50% mortality of nymphs tested was estimated at 2.19 × 104 spores/mL, and LC80 was 6.18 × 108 spores/mL, when bacterial spore suspensions were applied to citrus seedlings. Binding assays demonstrated that all Cry toxins present in strains S1302, S1450 and S1989 (Cry1Aa, Cry1Ab, Cry1Ac, Cry2Aa, Cry2Ab2, Cry1B and Cry11) interacted with the BBMV obtained from D. citri nymphs. In vivo assays showed that strains S1302 and S1450 were compatible with all pesticides commonly used in citrus orchards, althoug copper-based pesticides and dimethoate insecticide were incompatible in vitro with Bt strains. Our results demonstrate the potential of using Bt as systemic bioinsecticide in the future in HLB management.

O conhecimento dos eventos biológicos que ocorrem no periápice dos dentes com necrose pulpar se torna importante para compreender o desenvolvimento das lesões periapicais. Muitas são as moléculas e mediadores que participam na instalação da lesão periapical, a partir da infecção bacteriana que ocorre no interior dos canais radiculares. Assim, o objetivo do presente trabalho foi avaliar moléculas do sistema imune inato, da osteoclastogênese e metaloproteinases em lesões periapicais (LP) induzidas experimentalmente em camundongos knockout e wild type. Para esse objetivo, o presente estudo foi dividido em dois trabalhos distintos. O primeiro teve como objetivo avaliar a expressão de metaloproteinase 2 (MMP2) e metaloproteinase 9 (MMP9) durante a progressão da LP em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO), em comparação com camundongos wild type (WT). O segundo estudo avaliou a correlação da expressão gênica e imunomarcação de RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da LP em camundongos WT. No primeiro estudo lesões periapicais foram induzidas em molares inferiores de 54 camundongos TLR2 KO, MyD88 KO e WT (n=18/grupo). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. Os cortes histológicos foram submetidos a imunohistoquímica e posteriormente foi avaliada presença ou ausência de MMP2 e MMP9 nos diferentes grupos. No segundo estudo, 35 camundongos WT foram utilizados. As lesões periapicais foram induzidas nos primeiros molares inferiores de ambos os lados. Após 0 (G0), 7 (G7), 21 (G21) e 42 (G42) dias, os animais foram anestesiados e eutanasiados para que as mandíbulas fossem dissecadas e divididas ao meio.O lado direito das mandíbulas foi para o processamento histotécnico, para posterior marcação de RANK, RANKL, OPG, TLR2 e MyD88, por meio da imuno-histoquímica do lado esquerdo da mandíbula foi utilizado para a extração de RNA, para a determinação da expressão gênica de RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) e MyD88 (Myd88) utilizando quantificação em Tempo Real da Reação da Polimerase em Cadeia (qRT-PCR). Para ambos os estudos, testes paramétricos e não paramétricos foram realizados com nível de significância de 5%. Foi possível observar, no primeiro estudo, que nos períodos iniciais da progressão da lesão periapical, houve um aumento na imunomarcação de MMP9 nos camundongos TLR2 KO e MyD88 KO, quando comparados aos WT, diferente da MMP2 que não se observou nenhum aumento na imunomarcação. No entanto, aos 42 dias observou-se uma redução da imunomarcação de MMP2 e um aumento da MMP9 nos camundongos TLR2 KO. Adicionalmente, no segundo estudo, foi possível observar um aumento da imunomarcação para RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da lesão periapical (p<0,05). O aumento da expressão de Tnfrsf11 foi diferente entre os grupos G0 e G42, e G21 e G42 (p=0,006). No entanto, a expressão de Tnfrsf11b foi diferente entre os grupos G0 e G7, G7, G21 e G42, sendo possível observar uma diminuição dessa expressão ao longo do tempo (p<0,001). Tlr2 foi mais expresso entre os grupos G0 e G42 (p=0,03). E a expressão da molécula Myd88 foi estatisticamente significante entre os grupos G0 e G7, G21 e G42 (p=0,01). A razão Tnfrsf11/Tnfrsf11b aumentou durante a progressão da lesão periapical (p=0,002). Também foi possível observar uma correlação moderada entre Myd88 e Rankl (r=0,42; p=0,03) e entre Myd88 e Tlr2 (r=0,48; p<0,0001). Após as metodologias empregadas e os dados analisados, concluímos que a produção de MMP2 e MMP9 foi modulada por TLR2 e Myd88 durante a progressão da lesão periapical. Alem disso, podemos sugerir que existe uma correlação positiva entre o sistema RANK/RANKL/OPG e as proteínas do sistema imune inato, TLR2 e MyD88, durante a perda óssea decorrente da infecção bacteriana dos canais radiculares e posterior progressão da lesão periapical. / Knowledge of the biological events occurring inteeth apex with pulp necrosis becomes important to understand the development of periapical lesions. There are manymolecules and mediators that participate in the installation of the periapical lesion, from the bacterial infection that occurs inside the root canals. Thus, the aim of the present study was to evaluate molecules of the innate immune system, osteoclastogenesis and metalloproteinases in experimentally apical periodontitis (AP) induced in knockout and wild type mice. For this purpose, the present study was divided into two distinct studies. The first one aimed to evaluate the expression of metalloproteinases 2 (MMP2) and metalloproteinases 9 (MMP9) during the progression of AP in TLR2 knockout mice (TLR2 KO) and MyD88 knockout mice (MyD88 KO), compared to wild type mice (WT). The second study evaluated the correlation of gene expression and immunostaining of RANK, RANKL, OPG, TLR2 and MyD88 during LP progression in WT mice. In the first study AP were induced in lower molars of 54 TLR2 KO, MyD88 KO and WT mice (n = 18 / group). After 7, 21 and 42 days, the animals were euthanized and the jaws were dissected and submitted to histotechnical processing. The histological sections were submitted to immunohistochemistry and subsequently the presence or absence of MMP2 and MMP9 in the different groups was evaluated. In the second study, 35 WT mice were used. Periapical lesions were induced in the lower first molars on both sides. After 0 (G0) to 7 (G7), 21 (G21) and 42 (G42) days, the animals were anesthetized and euthanized so that the jaws were dissected and divided in half. The right side of the jaws was for the histotechnic processing, for subsequent imunostaining of RANK, RANKL, OPG, TLR2 and MyD88, through immunohistochemistry and the left side of the jaws was used for the extraction of RNA, for the determination of expression of RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) and MyD88 (Myd88) using Quantification Real Time of Polymerase Chain Reaction (qRT-PCR). For both studies, parametric and non-parametric tests were performed with significance level of 5%. It was possible to observe in the first study that in the initial periods of AP progression there was an increase in MMP9 immunostaining in TLR2 KO and MyD88 KO mice when compared to WT, different from MMP2 that no increase in immunostaining was observed. However, at 42 days there was a reduction in MMP2 immunostaining and an increase of MMP9 in TLR2 KO mice was observed. Additionally, in the second study, it was possible to observe an increase in the immunostaining for RANK, RANKL, OPG, TLR2 and MyD88 during periapical lesion progression (p <0.05). The increase in Tnfrsf11 expression was different between groups G0 and G42, and G21 and G42 (p = 0.006). However, the expression of Tnfrsf11b was different between the G0 and G7, G7, G21 and G42 groups, and a decrease in expression over time (p <0.001) was observed. Tlr2 was more expressed between the G0 and G42 groups (p = 0.03). And the expression of the Myd88 molecule was statistically significant between the G0 and G7, G21 and G42 groups (p = 0.01). The Tnfrsf11 / Tnfrsf11b ratio increased during the AP progression (p = 0.002). It was also possible to observe a moderate correlation between Myd88 and Rankl (r = 0.42, p = 0.03) and between Myd88 and Tlr2 (r = 0.48, p <0.0001). After the methodologies used and the data analyzed, we conclude that the production of MMP2 and MMP9 was modulated by TLR2 and Myd88 during the AP progression. In addition, we can suggest that there is a positive correlation between the RANK / RANKL / OPG system and the proteins of the innate immune system, TLR2 and MyD88, during bone loss due to bacterial infection of the root canals and subsequent progression of the apical periodontitis.

Much is known about how organs acquire their identity, yet we are only beginning to learn how their shape is regulated. Recent work has elucidated the role of coordinated cell division & expansion in determining plant organ shape. For instance, in Antirrhinum, leaf shape is affected in the cincinnata (cin) mutant because of an alteration in the cell division pattern. CIN codes for a TCP transcription factor and controls cell proliferation. It is unclear how exactly CIN-like genes regulate leaf morphogenesis. We have taken biochemical and genetic approach to understand the TCP function in general and the role of CIN-like genes in leaf morphogenesis in Antirrhinum and Arabidopsis. Targets of CINCINNATA To understand how CIN controls Antirrhinum leaf shape, we first determined the consensus target site of CIN as GTGGTCCC by carrying out RBSS assay. Mutating each of this target sequence, we determined the core binding sequence as TGGNCC. Hence, all potential direct targets of CIN are expected to contain a TGGNCC sequence. Earlier studies suggested that CIN activates certain target genes that in turn repress cell proliferation. To identify these targets, we compared global transcripts of WT and cin leaves by differential display PCR and have identified 18 unique, differentially expressed transcripts. To screen the entire repertoire of differentially expressed transcripts, we have carried out extensive micro-array analysis using 44K Arabidopsis chips as well as 13K custom-made Antirrhinum chips. Combining the RBSS data with the results obtained from the micro-array experiments, we identified several targets of CIN. In short, CIN controls expression of the differentiation-specific genes from tip to base in a gradient manner. In cin, such gradient is delayed, thereby delaying differentiation. We also find that gibberellic acid, cytokinin and auxin play important role in controlling leaf growth. Genetic characterization of CIN-homologues in Arabidopsis Arabidopsis has 24 TCP genes. Our work and reports from other groups have shown that TCP2, 4 and 10 are likely to be involved in leaf morphogenesis. These genes are controlled by a micro RNA miR319. To study the role of TCP4, the likely orthologue of CIN, we generated both stable and inducible RNAi lines. Down-regulation of TCP4 transcript resulted in crinkly leaves, establishing the role of TCP4 in leaf shape. To study the function of TCP2, 4 & 10 in more detail, we isolated insertion mutants in these loci. The strongest allele of TCP4 showed embryonic lethal phenotype, indicating a role for TCP4 in embryo growth. All other mutants showed mild effect on leaf shape, suggesting their redundant role. Therefore, we generated and studied various combinations of double and triple mutants to learn the concerted role of these genes on leaf morphogenesis. To further study the role of TCP4 in leaf development, we generated inducible RNAi and miRNA-resistant TCP4 transgenic lines and carried out studies with transient down-regulation and up-regulation of TCP4 function. Upon induction, leaf size increased in RNAi transgenic plants whereas reduced drastically in miR319 resistant lines, suggesting that both temporal & spatial regulation of TCP4 is required for leaf development. Biochemical characterization of TCP domain To study the DNA-binding properties of TCP4, random binding site selection assay (RBSS) was carried out and it was found that TCP4 binds to a consensus sequence of GTGGTCCC. By patmatch search and RT-PCR analysis, we have shown that one among 74 putative targets, EEL (a gene involved in embryo development), was down regulated in the RNAi lines of TCP4. This suggests that EEL could be the direct target of TCP4. We have tested this possibility in planta by generating transgenic lines in which GUS reporter gene is driven by EEL upstream region with either wild type or mutated TCP4 binding site. GUS analysis of embryos shows that transgenic with mutated upstream region had significantly reduced reporter activity in comparison to wild type, suggesting that EEL is a direct target of TCP4. We have further shown that TCP4 also binds to the upstream region of LOX2, a gene involved in Jasmonic acid (JA) biosynthesis (in collaboration with D. Weigel, MPI, Tubingen, Germany). TCP domain has a stretch of basic residues followed by a predicted helix-loop-helix region (bHLH), although it has little sequence homology with canonical bHLH proteins. This suggests that TCP is a novel and uncharacterized bHLH domain. We have characterized DNA-binding specificities of TCP4 domain. We show that TCP domain binds to the major groove of DNA with binding specificity comparable to that of bHLH proteins. We also show that helical structure is induced in the basic region upon DNA binding. To determine the amino acid residues important for DNA binding, we have generated point mutants of TCP domain that bind to the DNA with varied strength. Our analysis shows that the basic region is important for DNA binding whereas the helix-loop-helix region is involved in dimerization. Based on these results, we have generated a molecular model for TCP domain bound to DNA (in Collaboration with Prof. N. Srinivasan, IISc, Bangalore). This model was validated by further site-directed mutagenesis of key residues and in vitro assay. Functional analysis of TCP4 in budding yeast To assess TCP4 function in regulation of eukaryotic cell division, we have introduced TCP4 in S. cerevisiae under the GAL inducible promoter. TCP4 induction in yeast cells always slowed down its growth, indicative of its detrimental effect on yeast cell division. Flow cytometry analysis of synchronized cells revealed that TCP4 arrests yeast cell division specifically at G1→S boundary. Moreover, induced cells showed distorted cell morphology resembling shmoo phenotype. Shmooing is a developmental process which usually happened when the haploid cells get exposed to the cells of opposite mating type and get arrested at late G1 phase due to the inhibition of cdc28-cln2 complex. This suggested that TCP4-induced yeast cells are arrested at late G1 phase probably by the inhibition of cdc28-cln2 complex. To further investigate how TCP4 induce G1→S arrest, we carried out microarray analysis and found expression of several cell cycle markers significantly altered in TCP4-induced yeast cells. Studies on crinkly1, a novel leaf mutant in Arabidopsis To identify new genes involved in leaf morphogenesis, we have identified crinkly1 (crk1), a mutant where leaf shape and size are altered. We observed that crk1 also makes more number of leaves compared to wild type. Phenotypic analysis showed that crk1 leaf size is ~5 times smaller than that of wild type. Scanning electron microscopy (SEM) showed that both cell size and number are reduced in the mutant leaf, which explains its smaller size. We have mapped CRK1 within 3 cM on IV chromosome.

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Daktaro disertacijos „Kūginio pluošto kompiuterinės tomografijos diagnostinių galimybių endodontijoje tyrimai” tikslas - įvertinti kūginio pluošto kompiuterinės tomografijos (KPKT) galimybes tiriant viršūninio apydančio būklę ir danties šaknies kanalo gydymo rezultatus klinikinėmis sąlygomis bei ex vivo modelyje. Uždaviniai: 1) palyginti skaitmeninės intraoralinės rentgenografijos (SPR) ir KPKT metodų efektyvumą, diagnozuojant danties šaknies kanalo perforacijas žmogaus apatinio žandikaulio ex vivo modelyje bei nustatyti optimalų tūrinio vaizdo elemento dydį; 2) palyginti dantų šaknų perforacijų vietos ir dydžio įtaką KPKT diagnostinėms savybėms žmogaus apatinio žandikaulio ex vivo modelyje; 3) palyginti dantų šaknų perforacijų diagnozavimą skirtingose KPKT rekonstrukcinėse projekcijose žmogaus apatinio žandikaulio ex vivo modelyje; 4) palyginti SPR ir KPKT metodų tikslumą, vertinant endodontiškai gydytų pacientų dantų šaknų viršūninio apydančio (VA) būklę; 5) Sukurti pacientų VA būklės ir endodontinio gydymo kokybės įvertinimo sistemą (PESS), pagrįstą KPKT metodo tyrimo rezultatais; 6) Pagal kompleksinį viršūninio apydančio būklės įvertinimo indeksą, (COPI) ištirti pacientų VA būklę, naudojant KPKT ir skaitmeninę ortopantomografiją (SOR) bei palyginti abiem metodais gautus rezultatus; 7) Pagal endodontiškai gydytų dantų indeksą (ETTI), įvertinti pacientų endodontinio gydymo kokybę, naudojant KPKT ir SOR bei palyginti abiem metodais gautus rezultatus. / The aim of doctoral dissertation "Evaluation of diagnostic abilities of cone-beam computed tomography in endodontics” are to evaluate the cone-beam computed tomography (CBCT) diagnostic ability in assessment of periapical bone lesions and tooth root canal treatment outcome clinically and in ex vivo model. Objectives: 1) to compare the diagnostic abilities of CBCT and digital periapical radiogram (DPR) methods in the detection of root perforations ex vivo and to assess the use of different voxel sizes of a CBCT unit; 2) to assess the influence of root perforations sizes and locations on diagnostic performance of CBCT ex vivo; 3) to assess the influence of different reconstruction planes of CBCT in diagnosing root perforations ex vivo; 4) to compare the accuracy of DPR and CBCT in the detection of periapical radiolucencies in endodontically treated teeth; 5) to develop new patients Periapical and Endodontic Status Scale (PESS) by means of CBCT analysis; 6) to investigate periapical status of the patient using Complex Periapical Index (COPI) with two radiological methods (CBCT and digital orthopantomogram (DOR)) and to compare their results; 7) to investigate endodontic treatment quality using Endodontically Treated Tooth Index (ETTI) with two radiological methods (CBCT and DOR) and to compare their results.

O controle da extrusão apical durante a reintervenção endodôntica é essencial para o sucesso do novo tratamento. Nesse contexto, o presente estudo teve como objetivo comparar a quantidade de material sólido extruído na desobturação de canais radiculares variando-se a técnica de esvaziamento e o limite de obturação. Foram utilizados 40 incisivos inferiores previamente tratados divididos em dois grupos de acordo com o limite de obturação estabelecido. Cada grupo foi subdividido em dois subgrupos levando-se em conta a técnica de desobturação empregada; manual (subgrupos A1 e B1) e mecânico-rotatória com limas de Ni-Ti (Quantec LX) (subgrupos A2 e B2). O material sólido extruído foi coletado por meio do sistema de filtração Millipore, levado à secagem em dessecador de sílica e pesado em balança analítica de precisão. Os resultados obtidos foram submetidos a ANOVA para dois fatores de variação sendo em seguida empregado o Teste de Tukey (&#945; = 5%). A técnica de desobturação mecânico-rotatória produziu menor extrusão (0,66mg) que a manual (1,11mg), havendo diferença estatística significante entre elas (p < 0,05). Os canais preenchidos até o vértice radiográfico apresentaram maior quantidade de extrusão (1,38mg) do que os obturados 1 mm aquém do forame (0,39mg), observando-se diferença estatística significante entre eles (p < 0,05). A menor quantidade extrusão foi observada no subgrupo A2 (0,20mg), em que foi empregada a técnica rotatória de desobturação em canais obturados 1mm aquém do forame apical, sendo constatada diferença estatisticamente significante deste subgrupo com os demais (p < 0,05). A extrusão de material sólido durante a desobturação de canais radiculares é influenciado pela técnica empregada e pelo limite apical de obturação. / The apical extrusion control during the endodontic retreatment is essential for the success of the new treatment. The purpose of this study was to compare the quantity of solid apically extruded material during filling removal according the gutta-percha removal technique and root filling limit. Forty mandibular incisors with a single straight canal were selected. The canals were previously endodontically treated and then divided into two groups according the filling level. Each group was subdivided in two groups considering the retreatment technique: stainless steel hand files (subgroups A1 and B1) versus niquel-titanium rotatory instruments (subgroups A2 and B2). The extruded solid material was collected by Millipore filtration system, dried in silica desiccators and weighed in an eletrobalance. The results were analyzed using ANOVA with two variation factors and Tukey Test (&#945; = 5%). The niquel-titanium rotatory instruments produced less extrusion (0,66mg) than the stainless steel hand files (1,11mg), with significant statistical difference between them (p < 0,05). The canals filled until the radiographic apex showed larger amount of extruded material (1,38mg) than those filled 1 mm beyond the foramen (0,39mg). It was observed significant statistical difference between them (p < 0,05). The smaller extruded debris amount was observed in subgroup A2 (0,20mg), in which one the rotary technique was used to remove the gutta-percha of canals filled 1mm beyond the apical foramen. It was verified significant statistical difference of this subgroup with the other ones (p < 0,05). The extrusion of solid material during the gutta-percha removal is influenced by the technique as well as the apical filling limit.

O conhecimento dos eventos biológicos que ocorrem no periápice dos dentes com necrose pulpar se torna importante para compreender o desenvolvimento das lesões periapicais. Muitas são as moléculas e mediadores que participam na instalação da lesão periapical, a partir da infecção bacteriana que ocorre no interior dos canais radiculares. Assim, o objetivo do presente trabalho foi avaliar moléculas do sistema imune inato, da osteoclastogênese e metaloproteinases em lesões periapicais (LP) induzidas experimentalmente em camundongos knockout e wild type. Para esse objetivo, o presente estudo foi dividido em dois trabalhos distintos. O primeiro teve como objetivo avaliar a expressão de metaloproteinase 2 (MMP2) e metaloproteinase 9 (MMP9) durante a progressão da LP em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO), em comparação com camundongos wild type (WT). O segundo estudo avaliou a correlação da expressão gênica e imunomarcação de RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da LP em camundongos WT. No primeiro estudo lesões periapicais foram induzidas em molares inferiores de 54 camundongos TLR2 KO, MyD88 KO e WT (n=18/grupo). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. Os cortes histológicos foram submetidos a imunohistoquímica e posteriormente foi avaliada presença ou ausência de MMP2 e MMP9 nos diferentes grupos. No segundo estudo, 35 camundongos WT foram utilizados. As lesões periapicais foram induzidas nos primeiros molares inferiores de ambos os lados. Após 0 (G0), 7 (G7), 21 (G21) e 42 (G42) dias, os animais foram anestesiados e eutanasiados para que as mandíbulas fossem dissecadas e divididas ao meio.O lado direito das mandíbulas foi para o processamento histotécnico, para posterior marcação de RANK, RANKL, OPG, TLR2 e MyD88, por meio da imuno-histoquímica do lado esquerdo da mandíbula foi utilizado para a extração de RNA, para a determinação da expressão gênica de RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) e MyD88 (Myd88) utilizando quantificação em Tempo Real da Reação da Polimerase em Cadeia (qRT-PCR). Para ambos os estudos, testes paramétricos e não paramétricos foram realizados com nível de significância de 5%. Foi possível observar, no primeiro estudo, que nos períodos iniciais da progressão da lesão periapical, houve um aumento na imunomarcação de MMP9 nos camundongos TLR2 KO e MyD88 KO, quando comparados aos WT, diferente da MMP2 que não se observou nenhum aumento na imunomarcação. No entanto, aos 42 dias observou-se uma redução da imunomarcação de MMP2 e um aumento da MMP9 nos camundongos TLR2 KO. Adicionalmente, no segundo estudo, foi possível observar um aumento da imunomarcação para RANK, RANKL, OPG, TLR2 e MyD88 durante a progressão da lesão periapical (p<0,05). O aumento da expressão de Tnfrsf11 foi diferente entre os grupos G0 e G42, e G21 e G42 (p=0,006). No entanto, a expressão de Tnfrsf11b foi diferente entre os grupos G0 e G7, G7, G21 e G42, sendo possível observar uma diminuição dessa expressão ao longo do tempo (p<0,001). Tlr2 foi mais expresso entre os grupos G0 e G42 (p=0,03). E a expressão da molécula Myd88 foi estatisticamente significante entre os grupos G0 e G7, G21 e G42 (p=0,01). A razão Tnfrsf11/Tnfrsf11b aumentou durante a progressão da lesão periapical (p=0,002). Também foi possível observar uma correlação moderada entre Myd88 e Rankl (r=0,42; p=0,03) e entre Myd88 e Tlr2 (r=0,48; p<0,0001). Após as metodologias empregadas e os dados analisados, concluímos que a produção de MMP2 e MMP9 foi modulada por TLR2 e Myd88 durante a progressão da lesão periapical. Alem disso, podemos sugerir que existe uma correlação positiva entre o sistema RANK/RANKL/OPG e as proteínas do sistema imune inato, TLR2 e MyD88, durante a perda óssea decorrente da infecção bacteriana dos canais radiculares e posterior progressão da lesão periapical. / Knowledge of the biological events occurring inteeth apex with pulp necrosis becomes important to understand the development of periapical lesions. There are manymolecules and mediators that participate in the installation of the periapical lesion, from the bacterial infection that occurs inside the root canals. Thus, the aim of the present study was to evaluate molecules of the innate immune system, osteoclastogenesis and metalloproteinases in experimentally apical periodontitis (AP) induced in knockout and wild type mice. For this purpose, the present study was divided into two distinct studies. The first one aimed to evaluate the expression of metalloproteinases 2 (MMP2) and metalloproteinases 9 (MMP9) during the progression of AP in TLR2 knockout mice (TLR2 KO) and MyD88 knockout mice (MyD88 KO), compared to wild type mice (WT). The second study evaluated the correlation of gene expression and immunostaining of RANK, RANKL, OPG, TLR2 and MyD88 during LP progression in WT mice. In the first study AP were induced in lower molars of 54 TLR2 KO, MyD88 KO and WT mice (n = 18 / group). After 7, 21 and 42 days, the animals were euthanized and the jaws were dissected and submitted to histotechnical processing. The histological sections were submitted to immunohistochemistry and subsequently the presence or absence of MMP2 and MMP9 in the different groups was evaluated. In the second study, 35 WT mice were used. Periapical lesions were induced in the lower first molars on both sides. After 0 (G0) to 7 (G7), 21 (G21) and 42 (G42) days, the animals were anesthetized and euthanized so that the jaws were dissected and divided in half. The right side of the jaws was for the histotechnic processing, for subsequent imunostaining of RANK, RANKL, OPG, TLR2 and MyD88, through immunohistochemistry and the left side of the jaws was used for the extraction of RNA, for the determination of expression of RANK (Tnfrsf11a), RANKL (Tnfrsf11), OPG (Tnfrsf11b), TLR2 (Tlr2) and MyD88 (Myd88) using Quantification Real Time of Polymerase Chain Reaction (qRT-PCR). For both studies, parametric and non-parametric tests were performed with significance level of 5%. It was possible to observe in the first study that in the initial periods of AP progression there was an increase in MMP9 immunostaining in TLR2 KO and MyD88 KO mice when compared to WT, different from MMP2 that no increase in immunostaining was observed. However, at 42 days there was a reduction in MMP2 immunostaining and an increase of MMP9 in TLR2 KO mice was observed. Additionally, in the second study, it was possible to observe an increase in the immunostaining for RANK, RANKL, OPG, TLR2 and MyD88 during periapical lesion progression (p <0.05). The increase in Tnfrsf11 expression was different between groups G0 and G42, and G21 and G42 (p = 0.006). However, the expression of Tnfrsf11b was different between the G0 and G7, G7, G21 and G42 groups, and a decrease in expression over time (p <0.001) was observed. Tlr2 was more expressed between the G0 and G42 groups (p = 0.03). And the expression of the Myd88 molecule was statistically significant between the G0 and G7, G21 and G42 groups (p = 0.01). The Tnfrsf11 / Tnfrsf11b ratio increased during the AP progression (p = 0.002). It was also possible to observe a moderate correlation between Myd88 and Rankl (r = 0.42, p = 0.03) and between Myd88 and Tlr2 (r = 0.48, p <0.0001). After the methodologies used and the data analyzed, we conclude that the production of MMP2 and MMP9 was modulated by TLR2 and Myd88 during the AP progression. In addition, we can suggest that there is a positive correlation between the RANK / RANKL / OPG system and the proteins of the innate immune system, TLR2 and MyD88, during bone loss due to bacterial infection of the root canals and subsequent progression of the apical periodontitis.