L'hématopoïèse est le processus de formation des cellules sanguines différenciées à partir de cellules souches hématopoïétiques. Ce processus est étroitement contrôlé par l'intégration de signaux de développementaux et homéostatiques pour assurer une production équilibrée des différents types de cellules sanguines. Au niveau moléculaire, la régulation de ce processus est médiée par un certain nombre de facteurs de transcription, en particulier par les membres de la famille RUNX. Ainsi, des mutations affectant les membres de cette famille peuvent entrainer une déréglementation du programme de différenciation hématopoïétique et causer des hémopathies, dont des leucémies. D'une manière intrigante, de nombreux régulateurs de la transcription et des voies de signalisation contrôlant le développement des cellules sanguines sont évolutivement conservés des humains à Drosophila melanogaster, qui est donc utilisée comme organisme modèle pour étudier les mécanismes sous-jacents à la spécification des lignages sanguins et au contrôle de l'homéostasie des cellules sanguines. Les membres de la famille Myeloid Leukemia Factor (MLF) ont été impliqués dans l'hématopoïèse et dans la transformation oncogénique des cellules sanguines, mais leur fonction et leur mécanisme d'action moléculaire restent insaisissables. Des travaux précédents chez la Drosophile ont montré que MLF stabilise le facteur de transcription de type RUNX Lozenge (LZ) et contrôle le nombre de cellules sanguines LZ+. Au cours de ma thèse, j'ai cherché à déchiffrer le mécanisme moléculaire d'action de MLF sur Lozenge dans les cellules sanguines. Par une approche protéomique puis par des expériences de co-immunoprécipitation dans les cellules de Drosophile Kc167, nous avons identifié le co-chaperon de type Hsp40 DnaJ-1, et son partenaire le chaperon Hsc70-4, comme deux partenaires de MLF. De façon importante, nous avons montré que l'inhibition de l'expression de DnaJ-1 ou de Hsc70-4 dans les cellules Kc167 induit une réduction du niveau de protéine Lozenge et une diminution de sa capacité à activer la transcription très semblable à celles observées suite à l'inhibition de l'expression de MLF. De plus, la sur-expression de mutants de DnaJ-1 incapables d'activer le chaperon Hsc70-4 entraîne aussi une réduction du niveau de Lozenge et de sa capacité de transactivation et des expériences de coimmunoprécipitation montrent que Lozenge interagit avec MLF, DnaJ-1 et Hsc70-4. Nos résultats suggèrent donc que MLF agit au sein d'un complexe chaperon composé de DnaJ-1 et Hsc70-4 pour contrôler le niveau de Lozenge. En utilisant différents mutants de MLF ou DnaJ-1, nous avons montré que MLF et DnaJ-1 interagissent ensemble et avec Lozenge via des domaines phylogénétiquement conservés. D'autre part, des expériences de GST " pull down " in vitro suggèrent que ces trois protéines peuvent interagir ensemble directement. Nous proposons donc que MLF et DnaJ-1 contrôlent le niveau de protéine Lozenge en interagissant avec elle et en favorisant son repliement et/ou sa solubilité via l'activité chaperon de Hsc70-4. En parallèle, nous avons étudié la fonction de DnaJ-1 in vivo dans le développement des cellules sanguines de la Drosophile. Nos résultats montrent que, comme mlf, la perte de dnaj-1 s'accompagne d'une augmentation de la taille et du nombre des cellules sanguines LZ+, ainsi que d'une hyperactivation de la voie de signalisation Notch dans ces cellules. Nos résultats suggèrent que des hauts niveaux de Lozenge sont nécessaires pour contrôler le nombre et la taille des cellules LZ+ et pour inhiber l'expression de Notch. Nous proposons que le complexe MLF/DnaJ-1 contrôle le développement du lignage LZ+ en régulant le niveau de protéine Lozenge, et ainsi le niveau d'activité de la voie Notch. En conclusion, nos résultats ont mis à jour un lien fonctionnel entre MLF, le co-chaperon de type Hsp40 DnaJ-1 et un facteur de transcription de type RUNX, qui pourrait être conservé dans d'autres espèces. / Hematopoiesis is the process of formation of fully differentiated blood cells from hematopoietic stem cells (HSCs). This process is tightly controlled by the integration of developmental and homeostatic signals to ensure the generation of an appropriate number of each blood cell type. At the molecular level, the regulation of this developmental process is mediated by a number of transcription factors, especially by members of the RUNX family, and mutations affecting these factors are at the origin of numerous hemopathies, including leukemia. Intriguingly, many transcriptional regulators and signaling pathways controlling blood cell development are evolutionarily conserved from humans to Drosophila melanogaster. Hence, the fruit fly has become a potent and simplified model to study the mechanisms underlying the specification of blood cell lineages and the regulation of blood cell homeostasis. Members of the Myeloid Leukemia Factor (MLF) family have been implicated in hematopoiesis and in oncogenic blood cell transformation, but their function and molecular mechanism of action remain elusive. Previous work in Drosophila showed that MLF stabilizes the RUNX transcription factor Lozenge (LZ) and controls the number of LZ+ blood cells. During my PhD, I sought to further decipher the molecular mechanism of action of MLF on Lozenge during blood cell development. Using a proteomic approach in Drosophila Kc167 cells, we identified the Hsp40 co-chaperone family member DnaJ-1 and its chaperone partner Hsc70-4 as two partners of MLF. These interactions were confirmed by co-immunoprecipitations and in vitro pull-down assays. Importantly, we found that knocking down DnaJ-1 or Hsc70-4 expression in Kc167 cells caused a reduction in the level of Lozenge protein and a concomitant decrease in Lozenge transactivation activity, which were very similar to those caused by MLF knock-down. Similarly, over-expression of two DnaJ-1 mutants that are unable to stimulate the chaperone activity of Hsc70-4 also decreased Lozenge level and impaired its capacity to activate transcription. These results suggest that MLF could act within a chaperone complex composed of DnaJ-1 and Hsc70-4 to control Lozenge stability and activity. Along that line, we showed by co-immunoprecipitation that Lozenge interacts with MLF, DnaJ-1 and Hsc70-4, respectively. Using various truncated mutants of MLF or DnaJ-1, we showed that MLF and DnaJ-1 interact and together with Lozenge through their conserved MLF homology domain (MHD) and C-terminal region, respectively. Furthermore, in vitro GST pull-down assays suggested that the interactions between MLF, DnaJ-1 and Lozenge are direct. Thus, we propose that MLF and DnaJ-1 control Lozenge protein level by interacting with it and by promoting its folding and/or solubility via the Hsc70 chaperone machinery. In parallel, we assessed DnaJ-1 function in Drosophila blood cells in vivo using a null allele of dnaj-1 generated by CRISPR/Cas9 technique. We found that, like mlf, dnaj-1 mutation leads to an increase in the number and size of LZ+ blood cells, as well as to an over-activation of the Notch signaling pathway in these cells. Moreover, our data suggested that high levels of active Lozenge are required to control the number and size of LZ+ blood cells, and to down-regulate Notch expression. We propose that the MLF/DnaJ-1 complex controls LZ+ blood cell development in vivo by regulating Lozenge protein level/activity and thereby Notch pathway activation. In sum, our results establish a functional link between MLF, the Hsp40 co-chaperone DnaJ-1 and the RUNX transcription factor Lozenge, which could be conserved in other species.

In the UK, 12,000 patients die from metastatic breast cancer annually. There is therefore an urgent need to identify the molecules that cause metastasis. Recent work has revealed a role for the RUNX family of transcription factors in the development of metastatic breast cancer. The RUNX proteins form active transcription factor complexes when bound by the heterodimeric partner CBFβ to regulate the expression of metastatic genes. Previous work from our laboratory has demonstrated that knockdown of CBFβ resulted in a decreased invasion capacity of the metastatic breast cancer cell line MDA-MB-231. Three-dimensional culture of MDA-MB-231 cells revealed that loss of CBFβ induces a mesenchymal to epithelial transition (MET). The aim of this project was to determine the role of the RUNX/CBFβ complex in maintaining the mesenchymal phenotype of metastatic breast cancer cells. The data presented show that the phenotype changes were accompanied by changes in EMT marker-gene expression, including Snai2, MMP9, and MMP13. Induction of CBFβ in the CBFβ-knockdown cells remarkably restored both the invasive capacity and the mesenchymal phenotype. Further analysis revealed that maintenance of the mesenchymal phenotype was dependent upon both CBFβ-partner proteins, RUNX1 and RUNX2. Taken together the data presented in this thesis demonstrate that RUNX/CBFβ complexes drive the epithelial to mesenchymal transition (EMT) in breast cancer cells. These findings are likely to be important in the development of potential therapies to inhibit the metastatic spread of breast cancer.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24784号 / 医博第4976号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 藤田 恭之, 教授 髙折 晃史, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No / Runt-related (RUNX) transcription factors are evolutionarily conserved either in vertebrate or invertebrate. Lozenge (Lz), a members of RUNX family as well as homologue of AML-1, functions as an important transcription factor regulating the hemocytes differentiation. In this paper, we identified and characterized RUNX family especially Lz in silkworm, which is a lepidopteran model insect. The gene expression analysis illustrated that BmLz was highly expressed in hemocytes throughout the whole development period, and reached a peak in glutonous stage. Over-expression of BmLz in silkworm accelerated the melanization process of hemolymph, and led to instantaneously up-regulation of prophenoloxidases (PPOs), which were key enzymes in the melanization process. Further down-regulation of BmLz expression by RNA interference resulted in the significant delay of melanization reaction of hemolymph. These findings suggested that BmLz regulated the melanization process of hemolymph by inducing PPOs expression, and played a critical role in innate immunity defense in silkworm.

Introdução: A terapia com laser de baixa potência (LBP) vem sendo utilizada em Odontologia com diversos objetivos, em especial o de diminuir o tempo de cicatrização de feridas cirúrgicas. Objetivo: O objetivo do presente estudo in vivo foi avaliar qualitativamente e quantitativamente o efeito da irradiação com LBP no processo de remodelação óssea após a extração dentária em ratos jovens. Material e Método: Foram utilizados 60 ratos Wistar, distribuídos aleatoriamente nos seguintes grupos: Grupo Controle (n=30) animais com extração dentária sem aplicação do LBP, e Grupo Experimental (n=30) animais que tiveram extração dentária (Ext) e aplicação do LBP (Ext+LBP) nos três primeiros dias (54 J/cm2 por dia). Os animais foram sacrificados nos períodos de 1, 2, 3, 5, 7 e 10 dias após o procedimento de extração dentária. Neste estudo foram analisados os efeitos da aplicação do laser sobre o reparo alveolar através de microscopia de luz, luz polarizada e imunomarcação. Para isso foram avaliados os seguintes parâmetros: 1) porcentagem de formação óssea no interior do alvéolo; 2) grau de processo inflamatório; 3) grau de amadurecimento do colágeno; 4) imunomarcação para TRAP e RUNX-2. Todos os resultados obtidos foram submetidos à análise estatística através do teste ANOVA e teste de Mann-Whitney U (p<0.05). Resultados: Observou-se que os alvéolos do Grupo Experimental apresentaram processo de reparo mais evoluído quando comparados ao Grupo Controle, caracterizado pela organização mais rápida do coágulo sanguíneo, proliferação de fibroblastos nos restos do ligamento periodontal mais pronunciada, organização do colágeno e formação óssea mais precoce e mais intensa. Conclusão: A utilização do laser de baixa potência acelerou o processo de formação óssea durante as fases iniciais do experimento, embora nos períodos finais não houve diferença no processo de ossificação. / Introduction: Low Power Laser (LPL) therapy has been used in Dentistry to achieve many objectives, particularly to decrease the healing period on wound healing. Objectives: The purpose of this in vivo study was to evaluate qualitatively and quantitatively the irradiation effect with LPL in the bone remodeling process after tooth extraction in young rats. Material and Method: 60 Wistar rats were used, randomly distributed in the following groups: Control Group (n=30) animals with tooth extraction without LPL application, and Experimental Group (n=30) animals with tooth extraction (Ext) and LPL application (Ext+LPL) on the three fist days (54 J/cm2 per day). The animals were euthanized after the end periods of 1, 2, 3, 5, 7 and 10 days after the tooth extraction. This study looked into the effects of laser application on the alveolar repair through light microscopy, polarized light and immunostaining. After that, the following parameters were evaluated: 1) percentage of bone formation inside the dental alveolus; 2) degree of inflammatory process; 3) degree of collagen maturation; 4) immunostaining for TRAP and RUNX-2. All the results obtained were submitted to statistical analysis over the ANOVA and Mann- Whitney (p<0.05) test. Results: It was observed that the alveolus from the Experimental Group presented an improved repair process when compared to the Control Group, characterized by faster blood clot organization, more apparent fibroblasts proliferation on the remnants of the periodontal ligament, earlier and more intense collagen organization and bone generation. Conclusion: The use of Low Power Laser accelerated the bone generation process during the experimental initial period, although there was no difference in the ossification process in the final periods.

Introdução: A terapia com laser de baixa potência (LBP) vem sendo utilizada em Odontologia com diversos objetivos, em especial o de diminuir o tempo de cicatrização de feridas cirúrgicas. Objetivo: O objetivo do presente estudo in vivo foi avaliar qualitativamente e quantitativamente o efeito da irradiação com LBP no processo de remodelação óssea após a extração dentária em ratos jovens. Material e Método: Foram utilizados 60 ratos Wistar, distribuídos aleatoriamente nos seguintes grupos: Grupo Controle (n=30) animais com extração dentária sem aplicação do LBP, e Grupo Experimental (n=30) animais que tiveram extração dentária (Ext) e aplicação do LBP (Ext+LBP) nos três primeiros dias (54 J/cm2 por dia). Os animais foram sacrificados nos períodos de 1, 2, 3, 5, 7 e 10 dias após o procedimento de extração dentária. Neste estudo foram analisados os efeitos da aplicação do laser sobre o reparo alveolar através de microscopia de luz, luz polarizada e imunomarcação. Para isso foram avaliados os seguintes parâmetros: 1) porcentagem de formação óssea no interior do alvéolo; 2) grau de processo inflamatório; 3) grau de amadurecimento do colágeno; 4) imunomarcação para TRAP e RUNX-2. Todos os resultados obtidos foram submetidos à análise estatística através do teste ANOVA e teste de Mann-Whitney U (p<0.05). Resultados: Observou-se que os alvéolos do Grupo Experimental apresentaram processo de reparo mais evoluído quando comparados ao Grupo Controle, caracterizado pela organização mais rápida do coágulo sanguíneo, proliferação de fibroblastos nos restos do ligamento periodontal mais pronunciada, organização do colágeno e formação óssea mais precoce e mais intensa. Conclusão: A utilização do laser de baixa potência acelerou o processo de formação óssea durante as fases iniciais do experimento, embora nos períodos finais não houve diferença no processo de ossificação. / Introduction: Low Power Laser (LPL) therapy has been used in Dentistry to achieve many objectives, particularly to decrease the healing period on wound healing. Objectives: The purpose of this in vivo study was to evaluate qualitatively and quantitatively the irradiation effect with LPL in the bone remodeling process after tooth extraction in young rats. Material and Method: 60 Wistar rats were used, randomly distributed in the following groups: Control Group (n=30) animals with tooth extraction without LPL application, and Experimental Group (n=30) animals with tooth extraction (Ext) and LPL application (Ext+LPL) on the three fist days (54 J/cm2 per day). The animals were euthanized after the end periods of 1, 2, 3, 5, 7 and 10 days after the tooth extraction. This study looked into the effects of laser application on the alveolar repair through light microscopy, polarized light and immunostaining. After that, the following parameters were evaluated: 1) percentage of bone formation inside the dental alveolus; 2) degree of inflammatory process; 3) degree of collagen maturation; 4) immunostaining for TRAP and RUNX-2. All the results obtained were submitted to statistical analysis over the ANOVA and Mann- Whitney (p<0.05) test. Results: It was observed that the alveolus from the Experimental Group presented an improved repair process when compared to the Control Group, characterized by faster blood clot organization, more apparent fibroblasts proliferation on the remnants of the periodontal ligament, earlier and more intense collagen organization and bone generation. Conclusion: The use of Low Power Laser accelerated the bone generation process during the experimental initial period, although there was no difference in the ossification process in the final periods.

Objetivo: No atual trabalho propomo-nos a avaliar morfometricamente possíveis alterações na reparação óssea alveolar pós-exodontia de ratos tratados com Meloxicam, um anti-inflamatório inibidor preferencial da cicloxigenase 2 (COX-2) e correlacionar com a expressão temporal da COX-2 e do fator de transcrição 2 com domínio Runt (Runx-2) associada com a diferenciação de células da linhagem osteoblástica. Material e Métodos: A exodontia do incisivo superior direito foi realizada em 120 ratos Wistar, divididos em grupo controle (n = 60) - animais tratados com injeção intraperitoneal de 0,1 ml de solução salina 0,9% diariamente e grupo tratado (n = 60) animais tratados com injeção de Meloxicam na dose de 3mg/kg de massa corporal, diariamente, ambos durante 7 dias. O volume total do alvéolo (VtA) e do tecido ósseo (VtO), o número de células imunomarcadas/mm² (Nm) para COX-2 e Runx-2 e a expressão protéica por Western blotting (WB) da COX-2 e RUNX-2 foram avaliados nos períodos 3, 7, 10, 14, 21 e 30 dias póscirurgias. Resultados: No grupo tratado o VtA manteve-se constante até os 21 dias, enquanto que no controle foi 0,272 vezes menor em relação aos 3 dias decorrente da maior atividade osteoclástica. Porém, aos 14 dias, no grupo tratado o VtO foi 0,337 vezes menor em relação ao controle decorrente da inibição parcial da transmigração de células inflamatórias responsáveis pela degradação do coágulo e da angiogênese, ocasionando um retardo na formação dos tecidos de granulação/conjuntivo, na diferenciação das células osteoblásticas e na formação/remodelação do tecido ósseo, e consequentemente no reparo ósseo alveolar. A imunomarcação para COX-2 foi observada em diversos tipos celulares, como fibroblastos, células endoteliais, células inflamatórias, osteoblastos e osteócitos. O Nm para COX-2 não apresentou diferenças estatísticas significantes entre os grupos no intervalo de 3 e 21 dias pós-cirurgia, enquanto que, a expressão protéica pelo WB foi em média 0,232 vezes menor no grupo tratado em relação ao controle. Por outro lado, a imunomarcação para Runx-2 foi mais expressiva em osteoblastos e osteócitos e raramente em fibroblastos. O Nm para Runx-2 no grupo tratado durante todo período experimental foi em média 0,256 vezes menor em relação ao controle, sendo o mesmo observado para a expressão protéica pelo WB. Conclusão: Dentro dos limites da atual pesquisa, a aplicação do Meloxicam por um curto período de tempo atrasa temporariamente o processo inicial de reparo ósseo alveolar diminuindo a expressão das proteínas COX-2 e RUNX-2. / Objective: To evaluate morphometrically possible changes in post-extraction alveolar bone healing in rats treated with Meloxicam, a selective anti-inflammatory inhibitor of cyclooxygenase (COX-2) and to correlate it with the temporal expression of COX-2 and transcription factor 2 with Runt domain (Runx-2) associated with differentiation of osteoblastic lineage cells. Material and Methods: The extraction of the right upper incisor was made in 120 male Wistar rats, divided into control group (n=60) - animals treated with an intraperitoneal injection of 0,1 ml of 0,9% NaCl solution daily for 7 days and the treated group (n=60) - animals treated with injection of 3mg/kg of body weight of Meloxicam 0.9% NaCl solution daily for 7 days. The total alveolar volume (VtA), total bone tissue volume (VtO), number of immunohistochemically positive cells/mm² (Nm) for COX-2 and RUNX-2 and the Western blotting (WB) COX-2 and RUNX-2 protein expressions were evaluated after 3, 7, 10, 14, 21 and 30 days after the surgeries. Results: In the treated group the VtA remained constant until the 21st day, while in the control group at the same day the value was 0,272 times lower compared to the 3 days period, due to the higher osteoclastic activity. However, at 14 days the VtO was 0,337 times lower in the treated group compared to the control due to the partial inhibition of the transmigration of inflammatory cells responsible for the degradation of the clot and angiogenesis, causing a delay in the formation of granulation/connective tissues, differentiation of osteoblastic cells and in bone tissue formation/remodeling, and consequently in the alveolar bone repair. The immunostaining for COX-2 was observed in various cell types, such as fibroblasts, endothelial cells, inflammatory cells, osteoblasts and osteocytes. The Nm for COX-2 showed no statistical differences between groups from the 3rd to the 21st day, while the WB protein expression was on average 0,232 times lower in the treated group compared to the control. On the other hand, the immunostaining for Runx-2 was more expressive in osteoblasts and osteocytes and rarely in fibroblasts. The Nm for Runx-2 on the treated group was, during the whole experimental period, on average 0,256 times smaller than in the control. The same difference was observed in the protein expression by WB. Conclusion: Within the limits of the present study, its was concluded that the application of Meloxicam daily for 7 days causes a temporary delay in the alveolar bone repair decreasing the expression of proteins COX-2 and RUNX-2.

Objetivo: No atual trabalho propomo-nos a avaliar morfometricamente possíveis alterações na reparação óssea alveolar pós-exodontia de ratos tratados com Meloxicam, um anti-inflamatório inibidor preferencial da cicloxigenase 2 (COX-2) e correlacionar com a expressão temporal da COX-2 e do fator de transcrição 2 com domínio Runt (Runx-2) associada com a diferenciação de células da linhagem osteoblástica. Material e Métodos: A exodontia do incisivo superior direito foi realizada em 120 ratos Wistar, divididos em grupo controle (n = 60) - animais tratados com injeção intraperitoneal de 0,1 ml de solução salina 0,9% diariamente e grupo tratado (n = 60) animais tratados com injeção de Meloxicam na dose de 3mg/kg de massa corporal, diariamente, ambos durante 7 dias. O volume total do alvéolo (VtA) e do tecido ósseo (VtO), o número de células imunomarcadas/mm² (Nm) para COX-2 e Runx-2 e a expressão protéica por Western blotting (WB) da COX-2 e RUNX-2 foram avaliados nos períodos 3, 7, 10, 14, 21 e 30 dias póscirurgias. Resultados: No grupo tratado o VtA manteve-se constante até os 21 dias, enquanto que no controle foi 0,272 vezes menor em relação aos 3 dias decorrente da maior atividade osteoclástica. Porém, aos 14 dias, no grupo tratado o VtO foi 0,337 vezes menor em relação ao controle decorrente da inibição parcial da transmigração de células inflamatórias responsáveis pela degradação do coágulo e da angiogênese, ocasionando um retardo na formação dos tecidos de granulação/conjuntivo, na diferenciação das células osteoblásticas e na formação/remodelação do tecido ósseo, e consequentemente no reparo ósseo alveolar. A imunomarcação para COX-2 foi observada em diversos tipos celulares, como fibroblastos, células endoteliais, células inflamatórias, osteoblastos e osteócitos. O Nm para COX-2 não apresentou diferenças estatísticas significantes entre os grupos no intervalo de 3 e 21 dias pós-cirurgia, enquanto que, a expressão protéica pelo WB foi em média 0,232 vezes menor no grupo tratado em relação ao controle. Por outro lado, a imunomarcação para Runx-2 foi mais expressiva em osteoblastos e osteócitos e raramente em fibroblastos. O Nm para Runx-2 no grupo tratado durante todo período experimental foi em média 0,256 vezes menor em relação ao controle, sendo o mesmo observado para a expressão protéica pelo WB. Conclusão: Dentro dos limites da atual pesquisa, a aplicação do Meloxicam por um curto período de tempo atrasa temporariamente o processo inicial de reparo ósseo alveolar diminuindo a expressão das proteínas COX-2 e RUNX-2. / Objective: To evaluate morphometrically possible changes in post-extraction alveolar bone healing in rats treated with Meloxicam, a selective anti-inflammatory inhibitor of cyclooxygenase (COX-2) and to correlate it with the temporal expression of COX-2 and transcription factor 2 with Runt domain (Runx-2) associated with differentiation of osteoblastic lineage cells. Material and Methods: The extraction of the right upper incisor was made in 120 male Wistar rats, divided into control group (n=60) - animals treated with an intraperitoneal injection of 0,1 ml of 0,9% NaCl solution daily for 7 days and the treated group (n=60) - animals treated with injection of 3mg/kg of body weight of Meloxicam 0.9% NaCl solution daily for 7 days. The total alveolar volume (VtA), total bone tissue volume (VtO), number of immunohistochemically positive cells/mm² (Nm) for COX-2 and RUNX-2 and the Western blotting (WB) COX-2 and RUNX-2 protein expressions were evaluated after 3, 7, 10, 14, 21 and 30 days after the surgeries. Results: In the treated group the VtA remained constant until the 21st day, while in the control group at the same day the value was 0,272 times lower compared to the 3 days period, due to the higher osteoclastic activity. However, at 14 days the VtO was 0,337 times lower in the treated group compared to the control due to the partial inhibition of the transmigration of inflammatory cells responsible for the degradation of the clot and angiogenesis, causing a delay in the formation of granulation/connective tissues, differentiation of osteoblastic cells and in bone tissue formation/remodeling, and consequently in the alveolar bone repair. The immunostaining for COX-2 was observed in various cell types, such as fibroblasts, endothelial cells, inflammatory cells, osteoblasts and osteocytes. The Nm for COX-2 showed no statistical differences between groups from the 3rd to the 21st day, while the WB protein expression was on average 0,232 times lower in the treated group compared to the control. On the other hand, the immunostaining for Runx-2 was more expressive in osteoblasts and osteocytes and rarely in fibroblasts. The Nm for Runx-2 on the treated group was, during the whole experimental period, on average 0,256 times smaller than in the control. The same difference was observed in the protein expression by WB. Conclusion: Within the limits of the present study, its was concluded that the application of Meloxicam daily for 7 days causes a temporary delay in the alveolar bone repair decreasing the expression of proteins COX-2 and RUNX-2.

One of the primary goals of active research in molecular biology is to better understand the process of transcription regulation. An important objective in understanding transcription is identifying transcription factors that directly regulate target genes. Identifying these transcription factors is a key step toward eliminating genetic diseases or disease susceptibilities that are encoded inside deoxyribonucleic acid (DNA). There is much uncertainty and variation associated with transcription factor binding sites, requiring these sites to be represented stochastically. Although typically each transcription factor prefers to bind to a specific DNA word, it can bind to different variations of that DNA word. In order to model these uncertainties, we use a Bayesian approach that allows the binding probabilities associated with the motif to vary. This project presents a new method for motif searching that uses expert prior information to scan DNA sequences for multiple known motif binding sites as well as new motifs. The method uses a mixture model to model the motifs of interest where each motif is represented by a Multinomial distribution, and Dirichlet prior distributions are placed on each motif of interest. Expert prior information is given to search for known motifs and diffuse priors are used to search for new motifs. The posterior distribution of each motif is then sampled using Markov Chain Monte Carlo (MCMC) techniques and Gibbs sampling.

No description available.