Hydroxyproline (Hyp), a non-proteinogenic amino acid is a component of the organic material in bone. It has been used for 14C-dating of bone and the measurement of Hyp could be used as a biomarker in bone metabolism. Hydroxyproline is a component of collagen, the main structural protein in bone. The analyses of 14C in collagen and Hyp in human bones may provide timing information about bone processes and diseases, such as osteoarthritis and osteoporosis. The analysis of Hyp in bones (e.g., the determination of Hyp content) primarily relies on a spectrometric technique, liquid chromatography-mass spectrometry (LC-MS), and the determination of 14C content requires accelerator mass spectrometry (AMS). Moreover, to obtain these materials from bone requires the successful extraction of collagen and thr separation of Hyp from the collagen. This study aims at comparing methods for extracting collagen from bone, which do not destroy the Hyp. These methods include the use of either NaOH, KOH or HCl in one stage of the extraction process and separating sufficient Hyp for 14C analysis. This will provide information to determine whether Hyp can be used as a biomarker for bone diseases like osteoarthritis and osteoporosis. A preliminary 14C AMS analysis on collagen extracted by the NaOH method was carried out on human bones previously analyzed for forensic purposes. This demonstrated the ability of this technique to provide recent (post 1950) timing information. The collagen extractions by three different methods were first conducted on modern chicken bone, and the results showed that KOH method is the best bone collagen extraction method, yielding a largest quantity of Hyp. The KOH method was then employed to extract collagen from cow bone as a test of a more human-like (mammalian) material. As this was successful, collagen was extracted from diseased human bone fragments, obtained from the Ottawa Hospital. The data revealed that Hyp was successfully obtained from these bones. The study demonstrates that the extraction as well as the separation methods (preparative HPLC) can provide sufficient Hyp from bones for 14C AMS analysis. This will lead to future studies of Hyp in bone turnover, which may lead to its use as a novel biomarker for bone diseases such as osteoarthritis and osteoporosis.

Archaeological bones are usually dated by radiocarbon measurement of extracted collagen. However, low collagen content, contamination from the burial environment or museum conservation work have previously lead to inaccurate results, especially for old bones, compromising the ability to reconstruct reliable past chronologies. It is reported, for example, that up to 70% of Palaeolithic radiocarbon dates on bones are likely to be underestimates of the real age, blurring the picture of modern human dispersals and Neanderthal extinction. In this thesis, a method for isolating and radiocarbon dating the collagen amino acid hydroxyproline is described. Hydroxyproline consists of about 10% of bone collagen but is not found in significant amounts elsewhere in nature. The hydroxyproline dating method uses a mixed-mode (i.e. ion-exchange combined with hydrophobic chemistry), semi preparative HPLC methodology. The amino acids do not require derivatisation, and no organic solvents are used, thereby avoiding addition of carbon. The hypothesis of this thesis is that the hydroxyproline can be used as a bone specific biomarker, improving dating accuracy and making it possible to obtain radiocarbon determinations where previously it has been impossible. It was calculated that on average 3.3±1.4μg of contaminant carbon are added to each sample in the process of isolating the hydroxyproline, a low level suitable for 14C dating. It was investigated whether a deliberately contaminated bone and 'naturally' contaminated archaeological bones, yielding erroneous dates when dated using the normal pretreatment method, could be dated accurately using this method. In addition, a hydroxyproline date was obtained for a bone with too little surviving collagen to be dateable by the bulk collagen method. Finally, using the hydroxyproline dating method, the earliest direct ages for the presence of anatomically modern humans on the Russian Plain were obtained. The method proved to be a powerful tool that can help resolve longstanding archaeological questions.

As metaloproteinases da matriz (MMPs) são uma família de endopeptidades cálcio e zinco dependentes que participam da degradação de praticamente todos os componentes da matriz extracelular. Com o pressuposto de que estas enzimas podem estar relacionadas à progressão da erosão dental e que os agentes ácidos causadores da erosão podem influenciar na ativação das MMPs, o objetivo desse estudo in vitro foi avaliar a influência do pH sobre a atividade funcional das MMP-2 e -9 presentes na dentina coronária e radicular humana. O pó das dentinas coronária e radicular, provenientes de terceiros molares inclusos recém extraídos foi obtido separadamente e submetido ao protocolo de extração das proteínas, com ácido fosfórico a 1%. Após, o extrato contendo as proteínas e o pó de dentina parcialmente desmineralizado foram incubados em uma das respectivas soluções: solução 2 mM de APMA (acetato de 4-aminofenilmercúrio / grupo controle) ou em uma das soluções tampão (fosfato de potássio 0.1 M) com diferentes pHs (2.5, 4.5, 5.0, 6.0 e 7.0). Após a incubação, as proteínas foram separadas por eletroforese, em um gel de poliacrilamida copolimerizado com gelatina, para a avaliação da atividade gelatinolítica das MMPs, por zimografia. Esta análise foi realizada em triplicada e os zimogramas obtidos ao final foram avaliados por densitometria. A quantificação das bandas observadas nos zimogramas foi realizada pelo programa de imagens ImageJ e os dados avaliados de maneira descritiva e qualitativa. Para a avaliação do conteúdo de colágeno solubilizado, o pó de dentina parcialmente desmineralizado e incubado nos respectivos pHs (n = 8) foi mantido em um tampão de incubação durante 24h, e o conteúdo de hidroxiprolina (HYP) liberado neste meio foi mensurado em espectrofotômetro. Os dados obtidos foram avaliados estatisticamente por ANOVA 1 fator, seguido do teste de Tukey, com 5% de significância. A análise por zimografia mostrou bandas evidentes correspondente a MMP-2 na sua forma ativa (66kDa) e bandas menos expressivas relacionadas a sua forma latente (72kDa), tanto para a dentina coronária quanto para a radicular, em todos os grupos experimentais. Não foram identificadas bandas correspondentes a MMP-9, para nenhum dos substratos avaliados. As soluções com os menores pHs (2.5, 4.5 e 5.0) resultaram na maior atividade funcional da MMP-2, em comparação as soluções com os maiores pHs (6.0 e 7.0), em ambos os substratos. Para a análise de HYP, os grupos pH 2.5 e pH 4.5 mostraram valores de absorbância abaixo do limite de detecção do aparelho. Para os demais grupos experimentais, tanto para a dentina coronária quanto para a radicular, foram encontradas diferenças estatisticamente significantes entre eles (p < 0,05). O conteúdo de HYP liberada foi maior para o pH 7.0, comparado aos demais grupos (p < 0,05), exceto para o pH 6.0. Não foi encontrada diferença estatisticamente significante entre os grupos pH 6.0, pH 5.0 e controle (p > 0,05). Conclui-se que a atividade funcional da MMP-2 é dependente do pH. Os menores pHs promoveram um aumento na ativação da MMP-2 da dentina coronária e radicular humana. Nota-se em pHs próximos ao neutro, uma maior degradação da matriz orgânica dentinária desmineralizada por essas enzimas endógenas. / Matrix metalloproteinases (MMPs) are a family of endopeptidades calcium and zinc dependent that participate in the degradation of pratically all components of the extracellular matrix. With the assumption that these enzymes may be related to the progression of dental erosion and acidic agents that cause erosion can influence the activation of MMPs, the aim of this in vitro study was to evaluate the pH-influence on the functional activity of MMP-2 and -9 present in human coronal and root dentin. The powder of root and coronal dentine, from freshly extracted third molars was separately obtained and subjected to protein extraction protocol, with 1% phosphoric acid. After, the extract containing proteins and the partially demineralized powder were incubated in one of the following solutions: 2 mM APMA (4-aminophenylmercuric acetate / control) or one of the buffer solutions (0.1 M potassium phosphate) with different pHs (2.5, 4.5, 5.0, 6.0 and 7.0). After incubation, the proteins were separated by electrophoresis in a polyacrylamide gel copolymerized with gelatin, to evaluate the gelatinolytic activity of MMPs by means of zymography. This analysis was performed in triplicate and the zymograms obtained were evaluated by densitometry. Quantification of the bands observed in zymograms was performed by ImageJ software and the data were evaluated descriptively and qualitatively. To assess the solubilized dentin collagen, the partially demineralized dentin powder treated with different pHs (n=8) was incubated in an artificial saliva for 24 h, and the amount of hydroxyproline (HYP) released in media was measured by spectrophotometer. Data were statistically analyzed by ANOVA one factor, followed by the Tukey\'s test, with 5% significance. Zymography showed evident bands corresponding to active MMP-2 (66kDa) and less expressed bands related to its latent form (72kDa), for both coronal and root dentin, in all experimental groups. No bands corresponding to MMP-9 were identified, for both substrates. The lowest pH solutions (2.5, 4.5 and 5.0) yielded the higher functional activity than did the highest pH solutions (6.0 and 7.0), for both substrates. For HYP analysis, the groups pH 2.5 and pH 4.5 showed absorbance values below the detection limit of the equipment. For the other groups, for both coronal and root dentin, statistically significant diferences were found between them (p<0.05). The amount of HYP was higher for pH 7.0 than all other groups (p<0.05), except for pH 6.0. No statistical difference was found between pH 6.0, pH 5.0 and control (p>0.05). It can be concluded that the functional activity of MMP-2 is pH-dependent. Low pH solutions are able to increase the activation of human coronal and root MMP-2. It is noted in pHs close to neutral, a higher degradation of demineralized dentin organic matrix by these endogenous enzymes.

Made available in DSpace on 2014-06-11T19:31:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-04Bitstream added on 2014-06-13T20:01:37Z : No. of bitstreams: 1 pereira_rsc_dr_botfm.pdf: 7258621 bytes, checksum: 700f64c9060e3819c2ef6cd7d90e560c (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O consumo abusivo de álcool é considerado um grave problema de saúde pública mundial. O uso excessivo e indiscriminado de bebidas alcoólicas é responsável por mais de 60 agravos a saúde, como câncer de esôfago, estômago e fígado, doenças cardiovasculares, cirrose hepática, pancreatite crônica, úlcera péptica e em muitas dessas patologias pode-se necessitar de intervenções no trato digestivo. Na problemática exposta sobre o alcoolismo há uma série de alterações que podem repercutir nas anastomoses do trato gastrointestinal, como: desnutrição, lesões hepáticas, além do efeito tóxico direto do etanol sobre a mucosa gastrointestinal. Estudar o efeito do alcoolismo no processo de cicatrização intestinal e a evolução no pós-operatório de ratos submetidos à ingestão de etanol. Método: Foram utilizados 160 ratos da linhagem Wistar. Esses animais foram divididos em dois grupos, controle e tratado, sendo que o controle recebeu água e ração em livre demanda e o tratado solução etílica a 30% e ração livre demanda. Após 180 dias foi realizada colotomia cinco centímetros acima da deflexão peritoneal e anastomose em todos os animais. Após o procedimento os grupos foram divididos em quatro subgrupos de 20 ratos para estudo nos seguintes momentos: 40, 70, 140 e 210 pós-operatórios. Os parâmetros analisados foram: força de ruptura longitudinal, dosagem de hidroxiprolina tecidual, complicações pós-operatórias e estudo histopatológico. Resultados: O ganho de peso foi superior no grupo controle quando comparado com o grupo tratado (p<0,05). Agrupados todos os subgrupos, a força de ruptura foi significativamente maior no grupo controle que no grupo tratado (p<0,05). A dosagem de hidroxiprolina não apresentou diferença entre os grupos em cada momento estudado. A análise histopatológica não demonstrou alterações significativas entre... / Alcohol abuse is considered to be a serious public-health problem worldwide. The excessive and indiscriminate use of alcoholic beverages is responsible for more than 60 health medical problems, such as esophageal, stomach and liver cancer, cardiovascular diseases, hepatic cirrhosis, chronic pancreatitis and peptic ulcers. Many of these pathologies may require interventions in the digestive tract. The problems stemming from alcoholism include a number of alterations that may lead to anastomosis of the gastrointestinal tract, such as malnutrition and hepatic lesions, in addition to the direct toxic effect of ethanol on the gastrointestinal mucosa. Objective: To study the effect of alcoholism on the process of intestinal healing and the post-operative development of rats submitted to ethanol ingestion. Method: One hundred and sixty Wistar rats were used. These animals were divided into two groups, namely control and treated. The control group received water and animal feed ad libitum, and the treated group was given 30% ethyl alcohol solution and feed ad libitum. One hundred and eighty days later, colotomy 5 cm above peritoneal deflection and anastomosis were performed in all animals. After the procedure, the groups were divided into 4 sub-groups of 20 rats for study at the following post-operative moments: 4th, 7th, 14th and 21st days. The parameters analyzed were rupture strength, tissue hydroxyproline dosage, post-operative complications and histopathology. Results: Weight gain was greater in the control group as compared to that in the treated group (p<0.05). By grouping all the sub-groups, it was observed that rupture strength was significantly greater in the control group than in the treated group (p<0,05). Hydroxyproline dosage did not show any differences between the groups at each studied moment. Histopathological analysis did not show significant alterations between the groups... (Complete abstract click electronic access below)

Bidens pilosa L. is a traditional Chinese herbal medicine of which was considered as a potential COX2 inhibitor and anti-inflammatory agent. The objective of this study is to discriminate the protective effect of aqueous extract of Bidens pilosa L. leaves (BPLAE) against TAA-induced live fibrosis using an animal model. The herb extracts were administrated via intraperitoneal injection once per week (1.25, 2.5 g/kg), and thioacetamide (200 mg/kg) was injected three times per week and the mice were sacrificed at week 4 and week 8, respectively. Immunohistochemistry staining, Hematoxylin-eosin (HE) staining, Sirius red staining were carried out to evaluate the pathological alterations of mouse livers; in addition, Western blotting was performed to measure the differential expression of £\-smooth muscle actin (£\-SMA) between different treatment groups (vehicle, week 4 and week 8). Hepatic hydroxyproline was also detected in order to compare difference in collagen formation of each group. The results showed that Bidens pilosa L. effectively reduced amount of hepatic hydroxyproline and £\-SMA protein in mice with fibrotic liver induced by TAA. Moreover, in histiopathological exam, the BPLAE treated mice demonstrated a lower collagen and £\-SMA expression, which indicated that BPLAE might reduce degree and severity of liver fibrosis in mice. In conclusion, these results suggested that BPLAE potentially against fibrogenesis in TAA- induced mice liver fibrosis. Additionally, we found that BPLAE might involve in the signaling pathway of MAPK (ERK1/ERK2), which reduced the phosporylation level of p44 but not p42. Further studies using cell base assay to confirm the inhibiting role of BPLAE against cell proliferation or migration is warrant.

Submitted by Andrea Maselli (deamaselli@yahoo.com.br) on 2018-01-19T20:23:34Z No. of bitstreams: 2 Tese PDF Impressão.pdf: 1825558 bytes, checksum: 52a8a536c671378887d7f638e8244a82 (MD5) Tese PDF Impressão.pdf: 1825558 bytes, checksum: 52a8a536c671378887d7f638e8244a82 (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2018-01-23T19:31:17Z (GMT) No. of bitstreams: 1 maselli_a_me_sjc.pdf: 1719458 bytes, checksum: 9bbc488c5915b3ba270ef7fd508729ab (MD5) / Made available in DSpace on 2018-01-23T19:31:17Z (GMT). No. of bitstreams: 1 maselli_a_me_sjc.pdf: 1719458 bytes, checksum: 9bbc488c5915b3ba270ef7fd508729ab (MD5) Previous issue date: 2017-12-17 / A mensuração da degradação de colágeno radicular é um importante parâmetro para avaliar a progressão de cáries na dentina ou a eficácia de métodos terapêuticos na prevenção de lesões não cariosas. O objetivo deste trabalho foi avaliar a degradação do colágeno da dentina radicular frente a métodos de prevenção de cárie, utilizando o teste da Hidroxiprolina e a microradiografia. Foram obtidos 40 blocos de dentina radicular com aproximadamente 1,5 mm de profundidade x 6 mm de diâmetro, a partir de incisivos bovinos, os quais foram submetidos ao processo de desmineralização artificial em tampão acetato (pH=5), a fim de induzir a formação de uma lesão cariosa. Em seguida as amostras foram submetidas aos tratamentos preventivos de cárie radicular: 1) Clorexidina 0,12% 1 min, 2) Flúor neutro 2% 1 min, 3) Nd: YAG Laser- 60 mJ 10 s e 4) Água Deionizada (controle) 1 min. Após os tratamentos as amostras foram expostas à degradação pela enzima colagenase (Tipo VII, Produto No. C0773, Sigma-Aldrich, St. Louis, MO, USA) por um período de 5 dias. Ao final do desafio da colagenase, a solução contendo a enzima foi coletada para ser submetida ao teste da hidroxiprolina, para a mensuração da quantidade de colágeno degradado por meio de colorimetria em espectrofotômetro. Em seguida as mesmas amostras foram submetidas à mais 2 dias de desmineralização. Em complemento ao ensaio da hidroxiprolina as amostras foram expostas à microradiografia transversal para visualização e medição da área degradada. Uma análise descritiva dos dados obtidos foi realizada de modo a determinar o teste estatístico a ser aplicado. A análise de variância (ANOVA) para a HYP revelou que não houve diferença estatisticamente significativa entre os tratamentos empregados (p>0,05). Os dados obtidos na microradiografia foram submetidos ao teste de Kruskal Wallis e teste de comparações múltiplas, Dunn. Os dados de reptetição, comparações entre a desmineralização inicial (5 dias) e a segunda desmineralização (2 dias), foi realizado individulamente em cada grupo por teste T de medidas repetidas ou Wilcoxon. Houve diferença significativa entre os grupos (p<0,0001). Dos tratamentos empregados, a clorexidina e o flúor foram eficazes na prevenção da progressão da cárie radicular. / Quantification of collagen degradation is an important parameter to evaluate dentin caries progression of caries prevention aid. The aim of this study was to evaluate root collagen degradation against preventive methods by using the hydroxyproline assay and microradiography technique. Forty root dentin blocks were obtained with 1,5x6 mm (depth x diameter) from bovine incisors, which were submitted to artificial demineralization process by acetate buffer (PH=5), in order to induce a carious lesion. Then, the samples were submitted to preventive therapeutic treatment of root caries: 1) Chlorhexidine 0,12% 1 min, 2) Fluoride 2% 1 min, 3) Nd:YAG Laser - 60 mJ 10s, 4) Deionized Water (control) 1 min. After that, the samples were exposed to degradation by the collagenase enzyme (Type VII, Product No. C0773, Sigma- Aldrich, St. Louis, MO, USA) for 5 days. Following the collagenase challenge, the enzyme solution was collected for assaying hydroxyproline released from collgen matrix, where it is possilble to measure the amount of degraded collagen by colorimetry in a spectrophotometer. Soon after, the same samples were submitted to a further 2 days of demineralization process. In addition to the hydroxyproline assay the samples were exposed to the transverse microradiography for a visualization of the degraded area. Soon after the colorimetric test the same samples were submitted to further two days of demineralization. A descriptive analysis of the data will be performed to determine the statistical test to be applied. ANOVA test revealed that there was no difference between the treatments (p>0,05). The microradiography data were submitted to the Kruskal Wallis test and multiple comparison test, Dunn. The repetition data, comparisons between the initial (5 days) and the second (2 days) demineralization, were performed individually in each group by repeated measures T-test or Wilcoxon (p<0,0001). Among the proposed treatments, chlorhexidine and fluoride were effective in preventing root caries progression.

Made available in DSpace on 2014-06-11T19:23:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-04-20Bitstream added on 2014-06-13T20:30:37Z : No. of bitstreams: 1 teixeira_lg_me_jabo.pdf: 1248709 bytes, checksum: abf07304609e643fddd58d8b10e9193e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Avaliou-se por meio de análises clínico-laboratoriais e morfológicas, a evolução cicatricial de dois segmentos distintos do cólon descendente. Os equinos foram alocados em um único grupo e submetidos a três fases experimentais: obstrução experimental (F I), seguida de 13 dias pós-operatórios; anastomose término-terminal (F II) e videolaparoscopia (F III), ambas seguidas, cada uma, de seis dias pós-operatórios. O segmento distendido (SD) foi submetido a 240 minutos de distensão intraluminal, com pressão de 40 mmHg. O segmento controle (SC) não sofreu distensão. Colheram-se amostras do cólon descendente antes da obstrução (M0), após a desobstrução (M1) e durante a anastomose (M2). Realizou-se exame clínico antes de cada cirurgia (M0a, M0b e M0) e nos dias pós-operatórios (M12a-M312a, M24b-M120b e M24-M120). A colheita de sangue foi realizada antes de cada cirurgia (M0a, M0b e M0), durante a obstrução (M2a-M4a) e nos respectivos momentos pós-operatórios (M6a-M240a, M24b- M120b e M24-M120). Dois animais foram a óbito, sendo um durante F I e outro na F II. Verificou-se que a as alterações morfológicas e morfométricas intestinais foram mais evidentes na camada serosa no M2, no SD e SC, com muita semelhança entre os dois segmentos. Não houve diferença (p>0,5) na quantidade de colágeno determinada histologicamente e dosagem espectrofotométrica de hidroxiprolina. Durante a F II e F III, três e dois equinos, respectivamente, apresentaram aderências intestinais. Foram observadas diferenças (p>0,5) nos parâmetros clínicos e hematológicos apenas durante F I e F II. Concluiu-se que os equinos submetidos à obstrução experimental apresentaram pior prognóstico em relação à enteroanastomose... / The healing progression of two distinct segments of equine descending colon was evaluated by clinical, laboratorial and morphological analysis. Horses were allocated in only one group and submitted to three following experimental stages: experimental obstruction (F I) followed by thirteen days of postoperative care; end-toend anastomosis (F II) and the videolaparoscopy (F III), both last stages followed by six days of postoperative care. The distended segment (DS) underwent 40 mmHg intraluminal distension pressure for 240 minutes long. The control segment (CS) didn’t suffer any distension. Samples of the tissue of the descending colon was obtained before the obstruction (M0), after the desobstruction (M1) and during the anastomosis (M2). Clinical exam was done before each surgery (M0a, M0b and M0) and during the postoperative care days (M12a-M312a, M24b-M120b e M24-M120). Two animals died, one during F I and the other during F II. The intestinal morphological and morphometric changes were observed markedly on the serous layer on the M2 stage, on the DS and the CS, and it had similarity among the two segments. There was no difference (p>0,5) in the amount of collagen determined by histology and hydroxyproline spectrophotometric analysis. During F II and F III, three and two horses respectively, presented intestinal adhesions. Differences (p<0,5) in some of the clinical and hematological parameters were only observed during F I and F II. In conclusion, the horses that underwent experimental obstruction presented poor prognosis compared to the enteroanastomosis. The serialized clinical exams and the laboratory analyses provided good monitoring of the physiological immune responses during and after the intestinal obstruction or the anastomosis

Orientador: José Corrêa de Lacerda Neto / Banca: Rosemeri de Oliveira Vasconcelos / Banca: Rafael Resende Faleiros / Resumo: Avaliou-se por meio de análises clínico-laboratoriais e morfológicas, a evolução cicatricial de dois segmentos distintos do cólon descendente. Os equinos foram alocados em um único grupo e submetidos a três fases experimentais: obstrução experimental (F I), seguida de 13 dias pós-operatórios; anastomose término-terminal (F II) e videolaparoscopia (F III), ambas seguidas, cada uma, de seis dias pós-operatórios. O segmento distendido (SD) foi submetido a 240 minutos de distensão intraluminal, com pressão de 40 mmHg. O segmento controle (SC) não sofreu distensão. Colheram-se amostras do cólon descendente antes da obstrução (M0), após a desobstrução (M1) e durante a anastomose (M2). Realizou-se exame clínico antes de cada cirurgia (M0a, M0b e M0) e nos dias pós-operatórios (M12a-M312a, M24b-M120b e M24-M120). A colheita de sangue foi realizada antes de cada cirurgia (M0a, M0b e M0), durante a obstrução (M2a-M4a) e nos respectivos momentos pós-operatórios (M6a-M240a, M24b- M120b e M24-M120). Dois animais foram a óbito, sendo um durante F I e outro na F II. Verificou-se que a as alterações morfológicas e morfométricas intestinais foram mais evidentes na camada serosa no M2, no SD e SC, com muita semelhança entre os dois segmentos. Não houve diferença (p>0,5) na quantidade de colágeno determinada histologicamente e dosagem espectrofotométrica de hidroxiprolina. Durante a F II e F III, três e dois equinos, respectivamente, apresentaram aderências intestinais. Foram observadas diferenças (p>0,5) nos parâmetros clínicos e hematológicos apenas durante F I e F II. Concluiu-se que os equinos submetidos à obstrução experimental apresentaram pior prognóstico em relação à enteroanastomose ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The healing progression of two distinct segments of equine descending colon was evaluated by clinical, laboratorial and morphological analysis. Horses were allocated in only one group and submitted to three following experimental stages: experimental obstruction (F I) followed by thirteen days of postoperative care; end-toend anastomosis (F II) and the videolaparoscopy (F III), both last stages followed by six days of postoperative care. The distended segment (DS) underwent 40 mmHg intraluminal distension pressure for 240 minutes long. The control segment (CS) didn't suffer any distension. Samples of the tissue of the descending colon was obtained before the obstruction (M0), after the desobstruction (M1) and during the anastomosis (M2). Clinical exam was done before each surgery (M0a, M0b and M0) and during the postoperative care days (M12a-M312a, M24b-M120b e M24-M120). Two animals died, one during F I and the other during F II. The intestinal morphological and morphometric changes were observed markedly on the serous layer on the M2 stage, on the DS and the CS, and it had similarity among the two segments. There was no difference (p>0,5) in the amount of collagen determined by histology and hydroxyproline spectrophotometric analysis. During F II and F III, three and two horses respectively, presented intestinal adhesions. Differences (p<0,5) in some of the clinical and hematological parameters were only observed during F I and F II. In conclusion, the horses that underwent experimental obstruction presented poor prognosis compared to the enteroanastomosis. The serialized clinical exams and the laboratory analyses provided good monitoring of the physiological immune responses during and after the intestinal obstruction or the anastomosis / Mestre

As metaloproteinases da matriz (MMPs) são uma família de endopeptidades cálcio e zinco dependentes que participam da degradação de praticamente todos os componentes da matriz extracelular. Com o pressuposto de que estas enzimas podem estar relacionadas à progressão da erosão dental e que os agentes ácidos causadores da erosão podem influenciar na ativação das MMPs, o objetivo desse estudo in vitro foi avaliar a influência do pH sobre a atividade funcional das MMP-2 e -9 presentes na dentina coronária e radicular humana. O pó das dentinas coronária e radicular, provenientes de terceiros molares inclusos recém extraídos foi obtido separadamente e submetido ao protocolo de extração das proteínas, com ácido fosfórico a 1%. Após, o extrato contendo as proteínas e o pó de dentina parcialmente desmineralizado foram incubados em uma das respectivas soluções: solução 2 mM de APMA (acetato de 4-aminofenilmercúrio / grupo controle) ou em uma das soluções tampão (fosfato de potássio 0.1 M) com diferentes pHs (2.5, 4.5, 5.0, 6.0 e 7.0). Após a incubação, as proteínas foram separadas por eletroforese, em um gel de poliacrilamida copolimerizado com gelatina, para a avaliação da atividade gelatinolítica das MMPs, por zimografia. Esta análise foi realizada em triplicada e os zimogramas obtidos ao final foram avaliados por densitometria. A quantificação das bandas observadas nos zimogramas foi realizada pelo programa de imagens ImageJ e os dados avaliados de maneira descritiva e qualitativa. Para a avaliação do conteúdo de colágeno solubilizado, o pó de dentina parcialmente desmineralizado e incubado nos respectivos pHs (n = 8) foi mantido em um tampão de incubação durante 24h, e o conteúdo de hidroxiprolina (HYP) liberado neste meio foi mensurado em espectrofotômetro. Os dados obtidos foram avaliados estatisticamente por ANOVA 1 fator, seguido do teste de Tukey, com 5% de significância. A análise por zimografia mostrou bandas evidentes correspondente a MMP-2 na sua forma ativa (66kDa) e bandas menos expressivas relacionadas a sua forma latente (72kDa), tanto para a dentina coronária quanto para a radicular, em todos os grupos experimentais. Não foram identificadas bandas correspondentes a MMP-9, para nenhum dos substratos avaliados. As soluções com os menores pHs (2.5, 4.5 e 5.0) resultaram na maior atividade funcional da MMP-2, em comparação as soluções com os maiores pHs (6.0 e 7.0), em ambos os substratos. Para a análise de HYP, os grupos pH 2.5 e pH 4.5 mostraram valores de absorbância abaixo do limite de detecção do aparelho. Para os demais grupos experimentais, tanto para a dentina coronária quanto para a radicular, foram encontradas diferenças estatisticamente significantes entre eles (p < 0,05). O conteúdo de HYP liberada foi maior para o pH 7.0, comparado aos demais grupos (p < 0,05), exceto para o pH 6.0. Não foi encontrada diferença estatisticamente significante entre os grupos pH 6.0, pH 5.0 e controle (p > 0,05). Conclui-se que a atividade funcional da MMP-2 é dependente do pH. Os menores pHs promoveram um aumento na ativação da MMP-2 da dentina coronária e radicular humana. Nota-se em pHs próximos ao neutro, uma maior degradação da matriz orgânica dentinária desmineralizada por essas enzimas endógenas. / Matrix metalloproteinases (MMPs) are a family of endopeptidades calcium and zinc dependent that participate in the degradation of pratically all components of the extracellular matrix. With the assumption that these enzymes may be related to the progression of dental erosion and acidic agents that cause erosion can influence the activation of MMPs, the aim of this in vitro study was to evaluate the pH-influence on the functional activity of MMP-2 and -9 present in human coronal and root dentin. The powder of root and coronal dentine, from freshly extracted third molars was separately obtained and subjected to protein extraction protocol, with 1% phosphoric acid. After, the extract containing proteins and the partially demineralized powder were incubated in one of the following solutions: 2 mM APMA (4-aminophenylmercuric acetate / control) or one of the buffer solutions (0.1 M potassium phosphate) with different pHs (2.5, 4.5, 5.0, 6.0 and 7.0). After incubation, the proteins were separated by electrophoresis in a polyacrylamide gel copolymerized with gelatin, to evaluate the gelatinolytic activity of MMPs by means of zymography. This analysis was performed in triplicate and the zymograms obtained were evaluated by densitometry. Quantification of the bands observed in zymograms was performed by ImageJ software and the data were evaluated descriptively and qualitatively. To assess the solubilized dentin collagen, the partially demineralized dentin powder treated with different pHs (n=8) was incubated in an artificial saliva for 24 h, and the amount of hydroxyproline (HYP) released in media was measured by spectrophotometer. Data were statistically analyzed by ANOVA one factor, followed by the Tukey\'s test, with 5% significance. Zymography showed evident bands corresponding to active MMP-2 (66kDa) and less expressed bands related to its latent form (72kDa), for both coronal and root dentin, in all experimental groups. No bands corresponding to MMP-9 were identified, for both substrates. The lowest pH solutions (2.5, 4.5 and 5.0) yielded the higher functional activity than did the highest pH solutions (6.0 and 7.0), for both substrates. For HYP analysis, the groups pH 2.5 and pH 4.5 showed absorbance values below the detection limit of the equipment. For the other groups, for both coronal and root dentin, statistically significant diferences were found between them (p<0.05). The amount of HYP was higher for pH 7.0 than all other groups (p<0.05), except for pH 6.0. No statistical difference was found between pH 6.0, pH 5.0 and control (p>0.05). It can be concluded that the functional activity of MMP-2 is pH-dependent. Low pH solutions are able to increase the activation of human coronal and root MMP-2. It is noted in pHs close to neutral, a higher degradation of demineralized dentin organic matrix by these endogenous enzymes.

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