Imunoexpressão da proteína endonuclease apurínica/apurimidínica (APE-1) em adenomas pleomórficos e carcinomas ex-adenomas pleomórficos

Introduction: Apurinic/Apyrimidinic Endonuclease 1 (APE-1) is an essential protein for DNA base excision repair (BER) pathway and regulation of redox activities. The ability of malignant cells to recognize and repair DNA damage is an important mechanism for tumor survival, and recent studies suggest that APE-1 overexpression is related to poor prognosis in some tumors. Purpose: To analyze the immunoreactivity of APE-1 in Pleomorphic Adenomas (PA) and Carcinomas Ex Pleomorphic Adenomas (CaExPA) of salivary glands. Materials and Methods: A total of 49 tumors fixed in formalin and embedded in paraffin (33 PA and 16 CaExPA) underwent immunohistochemical study by the immunoperoxidase technique. APE-1 immunoreactivity was evaluated quantitatively by the percentage of immunopositive cells. For statistical analysis a significance level of 5% (p≤ 0.05) was adopted. Results: All cases of PA and CaExPA (n=49) were positive for APE-1, however, there was a higher expression in CaExPA, with statistically significant difference (p<0.001). There was no association between APE-1 expression and tumors of major or minor salivary gland, however, not encapsulated PA (median expression = 54.2%) showed higher expression when compared to encapsulated tumors (p=0.02). APE-1 overexpression was found mainly in cases of CaExAP with lymph node metastasis (median expression = 90.3% - p=0.002) and invasive pattern (median expression = 89.9% - p=0.003), when compared to cases without metastasis and intracapsular pattern. Conclusion: This study suggests that APE-1 is deregulated in the studied tumors. The increased expression of APE-1 is associated with the absence of complete capsule in PA and it is associated with more aggressive behavior in CaExPA.

Atividade antiangiogênica da forma recombinante da proteína 21 de Trypanosoma cruzi

Chagas disease, caused by the parasite Trypanosoma cruzi, is the cause of Chronic chagasic cardiomyopathy (CCC). The prospection of innovative therapeutic agents against CCC is a major task. The recombinant form of 21 (rP21), a secreted T. cruzi protein involved in host cell invasion and on progression of chronic inflammatory processes have been studied as a potential novel therapeutic target. Our present work aimed to verify and investigate the impact of rP21 in the formation of blood vessels in vitro and in vivo. First, tEnd cells were treated with different concentrations of rP21 or bacterial extract and viability and cellular adhesion were evaluated by MTT and angiogenesis inhibition by Matrigel tube formation assay and murine model. To verify the proteolytic activity of rP21 on extracellular matrix (ECM) components, fibrinogen, matrigel and fibronectin was incubated with rP21 or not. In addition, we performed proliferation assays and cell cycle analysis. Furthermore, the accumulation and distribution of F-actin was determined by Phalloidin staining using ImageJ software. Finally, tEnd cells were incubated with rP21 and the mRNA levels were analyzed by real-time PCR. Our results showed that rP21 did not alter cell viability and adhesion, but strongly inhibited vessel formation in vitro and in vivo. Tube formation assay showed that angiogenesis inhibition was dependent of the CXCR4-rP21 binding. In addition to these results, we observed that the rP21 was able to inhibit cell proliferation and promoted a significant reduction in the number of 4n cells (G2/M phase). Moreover, we found that rP21 significantly increased F-actin levels and this protein was able to modulate expression of genes related to angiogenesis and actin cytoskeleton. However, rP21 showed no significant activity on the matrix components. In this sense, we conclude that the rP21-endothelial cells (ECs) interaction via CXCR4 promotes inhibition of vessel formation through a cascade of intracellular events, such as inhibition of ECs proliferation and modulation of the expression of molecules associated with angiogenic processes and actin cytoskeleton.

Proteína C reactiva, índice de conicidad y factores de riesgo cardiovascular en pacientes con diabetes tipo 2

La Diabetes tipo 2, forma parte del clúster de componentes que integran el Síndrome metabólico, y constituye una enfermedad tremendamente prevalente en el mundo, con disfunciones metabólicas que incrementan la morbimortalidad. Objetivo. Con la finalidad de definir las características de una población amplia de pacientes diabéticos de la Comunidad de Madrid diagnosticados por el Hospital Infanta Leonor de Madrid, su contribución al síndrome metabólico, tipo de tratamiento, años de evolución y riesgo cardiovascular, se procedió a realizar un estudio transversal en una población de 735 diabéticos, seleccionados de una base de datos de 1135 diabéticos del Servicio de Endocrinología del Hospital de los que se disponían de datos sobre edad, género, parámetros antropométricos, glucosa, hemoglobina glicosilada, lípidos, lipoproteínas, consumo de tabaco, alcohol y actividad física. En muchos de ellos también se disponía de marcadores emergentes de afectaciones relacionadas con la diabetes y la enfermedad cardiovascular como PCR-us, microalbuminuria y fibrinógeno. Descripción de la muestra. La selección se realizó garantizando confidencialidad y que todos tuvieran datos de edad, sexo, y de los marcadores clásicos, y de la mayoría de los nuevos marcadores emergentes. Como quiera que contar absolutamente con todos los marcadores en el mismo individuo supondría perder en tal selección un número no despreciable de pacientes, se procedió a respetar la selección de 735 en donde 716 tenía información de la mayoría de los marcadores. A partir de los datos primarios se calcularon los cocientes colesterol/total /HDL-c, LDL-c/HDL-c que informan del riesgo cardiovascular, el cociente molar TG/HDL-c indicativo del tamaño de las LDL, y la relación de triglicérido- glucosa como marcador de resistencia a la insulina y riesgo de síndrome metabólico. Se procedió a calcular el riesgo cardiovascular según los algoritmos del estudio Framingham...

Valor de la proteína morfogenética 7 (BMP-7) en la artrodesis lumbar. Cohorte experimental con 7 años de seguimiento.

Se estudia la efectividad del factor de crecimiento BMP-7 en la artrodesis de columna lumbar. La efectividad se basa en la eficacia clínica, la seguridad y los resultados estudiados mediante imágenes radiográficas y de tomografía computadorizada concluyendo que la combinación de BMP-7 - aloinjerto es más efectiva que aloinjerto solo.

Modulação da expressão da proteína XPA em resposta ao tratamento com azul de metileno fotossensibilizado

Reactive oxygen species (ROS) are produced by aerobic metabolism and react with biomolecules, such as lipids, proteins and DNA. In high concentration, they lead to oxidative stress. Among ROS, singlet oxygen (1O2) is one of the main ROS involved in oxidative stress and is one of the most reactive forms of molecular oxygen. The exposure of some dyes, such as methylene blue (MB) to light (MB+VL), is able to generate 1O2 and it is the principle involved in photodynamic therapy (PDT). 1O2 e other ROS have caused toxic and carcinogenic effects and have been associated with ageing, neurodegenerative diseases and cancer. Oxidative DNA damage is mainly repaired by base excision repair (BER) pathway. However, recent studies have observed the involvement of nucleotide excision repair (NER) factors in the repair of this type of injury. One of these factors is the Xeroderma Pigmentosum Complementation Group A (XPA) protein, which acts with other proteins in DNA damage recognition and in the recruitment of other repair factors. Moreover, oxidative agents such as 1O2 can induce gene expression. In this context, this study aimed at evaluating the response of XPA-deficient cells after treatment with photosensitized MB. For this purpose, we analyzed the cell viability and occurrence of oxidative DNA damage in cells lines proficient and deficient in XPA after treatment with MB+VL, and evaluated the expression of this enzyme in proficient and complemented cells. Our results indicate an increased resistance to treatment of complemented cells and a higher level of oxidative damage in the deficient cell lines. Furthermore, the treatment was able to modulate the XPA expression up to 24 hours later. These results indicate a direct evidence for the involvement of NER enzymes in the repair of oxidative damage. Besides, a better understanding of the effects of PDT on the induction of gene expression could be provided

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Caracterização funcional de dois cDNAs homólogos à ciclofilina e à proteína reprimida por auxina (ARP) identificados em bibliotecas subtrativas a para floração em tomateiro

Flowering is a fundamental process in the life cycle for plant. This process is marked by vegetative to reproductive apical meristem conversion, due to interactions between several factors, both internal and external to plant. Therefore, eight subtractive libraries were constructed using apical meristem induced or not induced for two contrasting species: Solanum lycopersicum cv. Micro-Tom and Solanum pimpinellifolium. Several cDNAs were identified and among these, were selected two cDNAs: one homologous cDNA to cyclophilin (LeCYP1) and the other to Auxin repressed protein (ARP). It has observed that LeCYP1 and ARP genes are important in the developmental process to plants. In silico analysis, were used several databases with the exclusion criterion E-value <1.0x10-15. As a result, conservation was observed for proteins analyzed by means of multiple alignments and the presence of functional domains. Then, overexpression cassettes were constructed for the ARP cDNA in sense and antisense orientations. For this step, it was used the CaMV35S promoter. The cDNA orientation (sense or antisense) in relation to the promoter was determined by restriction enzymes and sequencing. Then, this cassette was transferred to binary vector pZP211 and these cassettes were transferred into Agrobacterium tumefaciens LBA4404. S. lycopersicum cv. Micro-Tom (MT) and MT-Rg1 plants were transformed. In addition, seedlings were subjected to hormone treatments using a synthetic auxin (- naphthalene acetic acid) and cyclosporin A (cyclophilin inhibitor) treatments and it was found that the hormone treatment there were changes in development of lateral roots pattern, probably related to decreases in auxin signaling caused by reduction of LeCYP1 in MT-dgt plants while cyclosporin A treatments, there was a slight delay in flowering in cv. MT plants. Furthermore, assay with real-time PCR (RT-qPCR) were done for expression level analysis from LeCYP1 and ARP in order to functionally characterize these sequences in tomato plants.

Estudo da proteína humana centrossomal TBCCD1 em condições de stress oxidativo causado por H2O2 em estado estacionário

Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz

Proteína C reactiva, marcador inflamatorio asociado con ANCA en tuberculosis pulmonar

Antecedentes: la proteína C reactiva es uno de los marcadores inflamatorios denominados “reactantes de fase aguda” que se produce en el hígado en respuesta a procesos infecciosos o inflamatorios. En los pacientes con tuberculosis se ha descrito la formación de anticuerpos anticitoplasma de neutrófi los (ANCA). Objetivo: determinar la concentración de proteína C reactiva, evaluar su comportamiento como marcador de la respuesta inflamatoria y analizar su correlación con los ANCA en los pacientes con tuberculosis pulmonar, antes y después de iniciar el tratamiento antifímico. Pacientes: se eligieron pacientes con sospecha de tuberculosis pulmonar. Una vez confirmado el diagnóstico, se obtuvieron las muestras de suero para analizar los datos clínicos y de laboratorios. La determinación de ANCA se realizó con estuches comerciales de inmunofluorescencia y la de proteína C reactiva con ELISA, antes y después de iniciar el tratamiento antifímico. Resultados: se obtuvieron 50 muestras de suero de pacientes con tuberculosis pulmonar. En la primera (94%) y segunda obtención (90%) de los sueros se registró un valor de proteína C reactiva menor de 5 mg/L. El valor promedio de proteína C reactiva fue de 3.05 ± 8.27 mg/L en la primera muestra y de 4.49 ± 11.2 mg/L en la segunda (p = 0.46). Los pacientes positivos a ANCA tuvieron valores más altos de proteína C reactiva en su segunda muestra (p = 0.001). Discusión: existe una asociación entre la proteína C reactiva y la producción de anticuerpos anticitoplasma de neutrófilos en un subgrupo de pacientes con tuberculosis pulmonar. Su significación es incierta, pero quizá desempeñan alguna función patogénica en la respuesta inflamatoria pulmonar.

Fraccionamiento de proteína de Bothriochloa saccharoides, Bothriochloa pertusa y Dichanthium annulatum

Gran parte de los sistemas de producción de rumiantes se encuentran ubicados en el trópico seco, donde las condiciones medioambientales son difíciles y la base de la alimentación son pastos que en su momento no aportan los niveles adecuados de proteína para el mantenimiento y producción de los animales. El objetivo de este trabajo fue determinar las fracciones nitrogenadas de Bothriochloa saccharoides, B. pertusa y Dichanthium annulatum comunes en el trópico seco del norte del Tolima, Colombia.

Estudo do papel da proteína multifuncional APE1/Ref-1 sobre a resposta inflamatória na meningite bacteriana

Despite advances in antibiotic therapy, bacterial meningitis (BM) remains with high mortality and morbidity rates in worldwide. One important mechanism associated to sequels during disease is the intense inflammatory response which promotes an oxidative burst and release of reactive oxygen species, consequently leading to cell death. Activation of DNA repair enzymes during oxidative stress has been demonstrated in several neurological disorders. APE1/Ref-1 is a multifunctional protein involved in DNA repair and plays a redox function on transcription factors such as NFkB and AP-1.The aim of this study was assess the role of APE1/Ref-1 on inflammatory response and the possibility of its modulation to reduce the sequels of the disease. Firstly it was performed an assay to measure cytokine in cerebrospinal fluid of patients with BM due to Streptococcus pneumoniae and Neisseriae meningitides. Further, a cellular model of inflammation was used to observe the effect of the inhibition of the endonuclease and redox activity of APE1/Ref-1 on cytokine levels. Additionally, APE1/Ref-1 expression in cortex and hippocampus of rat with MB after vitamin B6 treatment was evaluated. Altogether, results showed a similar profile of cytokines in the cerebrospinal fluid of patients from both pathogens, although IFNy showed higher expression in patients with BM caused by S. pneumoniae. On the other hand, inhibitors of APE1/Ref-1 reduced cytokine levels, mainly TNF-α. Reduction of oxidative stress markers was also observed after introduction of inhibitors in the LPS-stimulated cell. In the animal model, BM increased the expression of the protein APE1/Ref-1, while vitamin B6 promoted reduction. Thereby, this data rise important factors to be considered in pathogenesis of BM, e.g., IFNy can be used as prognostic factor during corticosteroid therapy, APE1/Ref-1 can be an important target to modulate the level of inflammation and VIII oxidative stress, and vitamin B6 seems modulates several proteins related to cell death. So, this study highlights a new understanding on the role of APE1/Ref-1 on the inflammation and the oxidative stress during inflammation condition

Proteína C reactiva y lactato deshidrogenasa en el diagnóstico de la obstrucción intestinal en un servicio de urgencias

Fundamento. La obstrucción intestinal es una de las urgencias quirúrgicas más frecuentes. Su diagnóstico se basa esencialmente en la historia clínica, la exploración física y las pruebas de imagen. El objetivo de este estudio fue analizar el valor diagnóstico de reactantes de fase aguda en en la obstrucción intestinal y discernir su naturaleza benigna versus maligna. Método. Se ha realizado un estudio de cohortes histórico sobre 53 pacientes sometidos a cirugía por obstrucción intestinal y/o cáncer colorrectal no obstructivo. Los pacientes se encuadraron en 3 grupos: grupo 1 (cáncer colorrectal con obstrucción intestinal) (n=23), grupo 2 (obstrucción intestinal benigna) (n=10) y grupo 3 (cáncer de colon no obstructivo) (n= 20). Se determinan los valores plasmáticos iniciales de la proteína C reactiva (PCR) y el enzima lactato deshidrogenasa (LDH). Resultados. La PCR se elevó cuantitativamente más en pacientes con obstrucción intestinal benigna (grupo 2) (p=0,001), mientras que la LDH en el grupo 1 (pacientes con cáncer obstructivo). Los niveles plasmáticos de LDH fueron significativamente mayores en los grupos con obstrucción intestinal (Grupos 1 y 2) que en pacientes sin obstrucción (p<0,001). Niveles plasmáticos de PCR mayores de 11 mg/l y de LDH mayores de 317 U/L mostraron una validez diagnóstica aceptable para diferenciar los pacientes con obstrucción intestinal, con áreas bajo la curva ROC de 80% (IC 95% = 68-92%) y de 86% (IC 95%= 75-96%) respectivamente. Su validez diagnóstica para diferenciar el origen benigno o maligno es menor, con áreas bajo la curva ROC de 56% para niveles de PCR > 24 ng/l (IC 95% = 30-82%) y de 52% (IC 95% = 29-74%) para niveles de LDH > 359 U/L. Conclusión. La determinación de las concentraciones plasmáticas de la PCR y la LDH pueden ayudar al diagnóstico de la obstrucción intestinal y orientar hacia su origen benigno o maligno en los servicios de urgencias.

Efeito do nível de proteína da ração no crescimento de Tenca (Tinca tinca), no crescimento de manjerição (Ocimum basilicum) e no balanço de nutrientes num sistema de Aquaponia

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014