Avaliação do efeito do composto tipo heparina isolado do caranguejo Chaceon fenneri na hemostasia e na morte celular

Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents

Inflamación celular en pacientes sometidos a cirugía electiva tratados con carbohidratos de baja carga glicémica, ácidos grasos omega 3 y antioxidantes

Antecedentes y Objetivos. La creciente complejidad de la Especialidad de Cirugía Plástica, ha inducido a su ramificación en varias subespecialidades. Una de las limitantes de la Cirugía Plástica ha sido la obesidad, enfermedad que conlleva complicaciones, aumenta el riesgo de inconvenientes y hace insuficiente el resultado proyectado para la estética y la función. El objetivo del presente trabajo es mostrar la importancia de la evaluación de la inflamación celular en la preparación de pacientes sometidos a cirugía electiva, previamente tratados con dieta con carbohidratos de baja carga glicémica, ácidos grasos omega 3 y 6 y antioxidantes. Material y Método. Realizamos un estudio longitudinal prospectivo cuasi-experimental no aleatorio de 23 pacientes que solicitaron intervenciones de Cirugía Plástica y aceptaron entrar en el protocolo de diagnóstico y tratamiento de inflamación celular. Primero realizamos el cuestionario de Reporte de Inflamación Celular "RIS" y tomamos a los pacientes pruebas de inflamación celular y de ácido eicosapentaenóico (EPA), ácido decosaexaenóico (DHA), acido araquidónico (AA) y ácido dihomogamalinoléico (DGLA) al ingreso y antes de la cirugía. Analizamos los datos con estadística descriptiva y comparamos los rangos con la prueba McNemar y de hipótesis t de student, del sistema SPSS. Resultados. Tras aplicar el RIS a los 23 pacientes antes de la preparación con dieta y tras un periodo de 1 a 3 meses, evaluamos las respuestas de cada uno de los cuestionarios a través de las pruebas no paramétricas, encontrando diferencia significativa en los 14 items. Se mejoró considerablemente a la alza el EPA; el DHA y el AA sin cambios significativos; y el DGLA disminuyó considerablemente. Con t de student encontramos variación significativa en los fosfolípidos del plasma y no hubo diferencia significativa entre los DHA y AA. Conclusiones. Demostramos así la efectividad de la dieta de los omegas, que contribuye a la disminución de la sintomatología inicial de los pacientes.

Influência da suplementação de retinol palmitato sobre os níveis de vitamina A no leite de puérperas saudáveis

The tendency towards reduction of serum retinol levels, an existing placental barrier and the increase of retinol demand, are factors that place puerperal and lactating women at risk for Vitamin A deficiency. This micronutrient is an essential component of vital processes such as differentiation, cellular proliferation, and apoptosis. The objective of this study is to evaluate the effect of palmitate retinol supplementation (100.000UI) upon the milk retinollevels in puerperal women at the Januário Cicco University Maternity Hospital. This intervention has been adopted by the Ministry of Health since 2002. The longitudinal experiment was conducted with 106 puerperal women (68 comprised the supplemented group and 38 the control group). The High Performance Liquid Chromatography (HPLC) method was used to dose the retinol of the milk and serum samples, and the creamtocrit method to determine the milk fat levels. The retinol means for the colostrums were 99.0 ± 64.4 ug/dL and 160.1 ± 94,4 ug/dl 6 hours afier supplementation; 68.9 ± 33.5 ug/dL for the transitional milk, and 30.6 ± 15.2 ug/dL for the mature milk of the supplemented group. Ali the difterences between means were statistically significant. The difterence between retinol means in the control group were also significant, with these being greater in the colostrum, 88.6 ± 62.1 ug/dL with 61.9 ± 30.1 ug/dl in the transition milk and 32.9 ±32.9 ± 17.6 ug/dL in the mature milk. No significant difference was observed in the retinol means of the three types ot milk in the supplemented group when compared to their respective means in the control group. The prevalence in serum (35.1 % and 81.1 % for the cutting point 20 ug/dL, respectively) and in milk (51.4%) revealed vitamin A deficiency as a public health problem. COlostrum, transition, and mature milk tats varied similarly in the supplemented group (1,92 ± 0,96; 3,25 ± 1,27 and 3,31 ± 1,36 grams) and in the control group (1,87 ± 1,14; 3,25 ± 1,31 and 3,36 ± 1,67 grams), with an observed difference between the colostrum/transition milk and the colostrum/mature milk fats. No difference was observed between the groups. The study showed that the 200.000UI supplementation was not sufficient to increase the milk retinol to the desired levels nor to meet the demands of the mothers with deprived hepatic reserves. It is suggested that another similar dose be offered within 30 days or less, and within 2 months post-partum, while continual/y monitoring for possible pregnancy

Análise da estabilidade genética de células-tronco mesenquimais humanas

Human multipotent mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have become an important and attractive therapeutic tool since they are easily isolated and cultured, have in vitro expansion potential, substantial plasticity and secrete bioactive molecules that exert trophic effects. The human umbilical cord as a cell source for cell therapy will help to avoid several ethical, political, religious and technical issues. One of the main issues with SC lines from different sources, mainly those of embryonic origin, is the possibility of chromosomal alterations and genomic instability during in vitro expansion. Cells isolated from one umbilical cord exhibited a rare balanced paracentric inversion, likely a cytogenetic constitutional alteration, karyotype: 46,XY,inv(3)(p13p25~26). Important genes related to cancer predisposition and others involved in DNA repair are located in 3p25~26. Titanium is an excellent biomaterial for bone-implant integration; however, the use can result in the generation of particulate debris that can accumulate in the tissues adjacent to the prosthesis, in the local bone marrow, in the lymph nodes, liver and spleen. Subsequently may elicit important biological responses that aren´t well studied. In this work, we have studied the genetic stability of MSC isolated from the umbilical cord vein during in vitro expansion, after the cryopreservation, and under different concentrations and time of exposition to titanium microparticles. Cells were isolated, in vitro expanded, demonstrated capacity for osteogenic, adipogenic and chondrogenic differentiation and were evaluated using flow cytometry, so they met the minimum requirements for characterization as MSCs. The cells were expanded under different concentrations and time of exposition to titanium microparticles. The genetic stability of MSCs was assessed by cytogenetic analysis, fluorescence in situ hybridization (FISH) and analysis of micronucleus and other nuclear alterations (CBMN). The cells were able to internalize the titanium microparticles, but MSCs preserve their morphology, differentiation capacity and surface marker expression profiles. Furthermore, there was an increase in the genomic instability after long time of in vitro expansion, and this instability was greater when cells were exposed to high doses of titanium microparticles that induced oxidative stress. It is necessary always assess the risks/ benefits of using titanium in tissue therapy involving MSCs, considering the biosafety of the use of bone regeneration using titanium and MSCs. Even without using titanium, it is important that the therapeutic use of such cells is based on analyzes that ensure quality, security and cellular stability, with the standardization of quality control programs appropriate. In conclusion, it is suggested that cytogenetic analysis, FISH analysis and the micronucleus and other nuclear alterations are carried out in CTMH before implanting in a patient

Evaluación de la expresión de enzimas hidrolíticas de pared celular en cloroplastos de tabaco

La alta capacidad del genoma del cloroplasto para integrar y expresar transgenes en altos niveles, hace de la tecnología transplastómica una buena opción para producir proteínas de interés. Este reporte presenta la expresión estable de una pectinasa (gen PelA), una β-glucosidasa (gen Bgl1), dos celulasas (genes CelA y CelB) y la primer expresión estable de una manganeso peroxidasa (gen MnP-2) en el genoma de cloroplastos de tabaco. Se construyeron seis vectores: pES4, pES5, pES6, pHM4, pHM5 y pHM6 derivados de pPRV111A conteniendo los genes sintéticos PelA, MnP- 2, Bgl1, CelA-CelB, CelA y CelB, respectivamente. Los genes se flanquearon por un promotor sintético del gen rrn16S y una secuencia sintética 3’UTR del gen rbcL. La integración en la región intergénica rrn16S y 3'rps12 se confirmó por análisis de Southern blot. El procesamiento estable de los transcritos se confirmó por un análisis de Northern blot. Se realizó un análisis enzimático para detectar la expresión y funcionalidad de las enzimas recombinantes, las plantas maduras mostraron mayor actividad comparado con plantas de tipo silvestre. Las plantas transplastómicas exhibieron 58.5% más actividad de pectinasa a pH neutro y a 60°C, mientras que manganeso peroxidasa mostró alta actividad a pH 6 y 65°C; en el caso de las celulasas, todas las enzimas mostraron mayor actividad a pH 5 (β-glucosidasa: 30.45 xviii U/mg, CelA-CelB 58 U/mg, CelA 49.10 U/mg y CelB 48.72 U/mg) a 40°C para β- glucosidasa y 65°C para celulasas. Las plantas transplastómicas mostraron un desarrollo similar a las plantas de tipo silvestre; sin embargo, la línea pHM4 mostró fenotipos variegados en hojas. Los análisis mostraron que los genes de enzimas hidrolíticas PelA, MnP-2, Bgl1, CelA-CelB, CelA y CelB pueden integrarse y expresarse en el genoma de cloroplastos con alta actividad; de este modo, debido a que una planta madura en promedio cuenta con ~ 470 g de biomasa, es posible producir 66,676.25 unidades de pectinasa, 21,715.46 unidades de manganeso peroxidasa, 338,081.0 unidades de celulasas A-B, 231,456.7 unidades de celulasa A, 206,669.8 unidades de celulasa B y 139,395.0 unidades de β-glucosidasa por planta. Este estudio sustenta información sobre métodos y estrategias de expresión de enzimas hidrolíticas con potencial aplicación biotecnológica utilizando plantas transplastómicas.

Transcriptoma diferencial entre células-tronco mesenquimais humanas jovens e senescentes

Human mesenchymal stem cells (MSC) are powerful sources for cell therapy in regenerative medicine. The long time cultivation can result in replicative senescence or can be related to the emergence of chromosomal alterations responsible for the acquisition of tumorigenesis features in vitro. In this study, for the first time, the expression profile of MSC with a paracentric chromosomal inversion (MSC/inv) was compared to normal karyotype (MSC/n) in early and late passages. Furthermore, we compared the transcriptome of each MSC in early passages with late passages. MSC used in this study were obtained from the umbilical vein of three donors, two MSC/n and one MSC/inv. After their cryopreservation, they have been expanded in vitro until reached senescence. Total RNA was extracted using the RNeasy mini kit (Qiagen) and marked with the GeneChip ® 3 IVT Express Kit (Affymetrix Inc.). Subsequently, the fragmented aRNA was hybridized on the microarranjo Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix Inc.). The statistical analysis of differential gene expression was performed between groups MSC by the Partek Genomic Suite software, version 6.4 (Partek Inc.). Was considered statistically significant differences in expression to p-value Bonferroni correction ˂.01. Only signals with fold change ˃ 3.0 were included in the list of differentially expressed. Differences in gene expression data obtained from microarrays were confirmed by Real Time RT-PCR. For the interpretation of biological expression data were used: IPA (Ingenuity Systems) for analysis enrichment functions, the STRING 9.0 for construction of network interactions; Cytoscape 2.8 to the network visualization and analysis bottlenecks with the aid of the GraphPad Prism 5.0 software. BiNGO Cytoscape pluggin was used to access overrepresentation of Gene Ontology categories in Biological Networks. The comparison between senescent and young at each group of MSC has shown that there is a difference in the expression parttern, being higher in the senescent MSC/inv group. The results also showed difference in expression profiles between the MSC/inv versus MSC/n, being greater when they are senescent. New networks were identified for genes related to the response of two of MSC over cultivation time. Were also identified genes that can coordinate functional categories over represented at networks, such as CXCL12, SFRP1, xvi EGF, SPP1, MMP1 e THBS1. The biological interpretation of these data suggests that the population of MSC/inv has different constitutional characteristics, related to their potential for differentiation, proliferation and response to stimuli, responsible for a distinct process of replicative senescence in MSC/inv compared to MSC/n. The genes identified in this study are candidates for biomarkers of cellular senescence in MSC, but their functional relevance in this process should be evaluated in additional in vitro and/or in vivo assays

Prática de ensino supervisionada em educação pré-escolar e 1º ciclo do ensino básico: ensinar para aprender - gerindo a heterogeneidade, o currículo e rumando à diferenciação pedagógica

O presente relatório surge no âmbito do Mestrado em Educação Pré-escolar e Ensino do 1º Ciclo do Ensino Básico e visa a obtenção do grau de mestre nos níveis de escolaridade referidos. Neste descrevo o projeto de investigação-ação desenvolvido nos contextos da Prática de Ensino Supervisionado (PES) em Educação Pré-escolar e 1º Ciclo do Ensino Básico (CEB). A investigação-ação realizada visava, sobretudo, ajudar-me a compreender quais as condições que deveria reunir de modo a responder de forma adequada às necessidades das crianças e a perceber como poderia melhorar a minha ação e intervenção promovendo aprendizagens significativas e de qualidade, salientando que o propósito não era fornecer o mesmo a todas as crianças, mas sim, a cada uma aquilo de que necessitava. A minha aposta na Diferenciação Pedagógica e numa gestão flexível do currículo surge orientada pelo princípio do direito de todos à aprendizagem e como resposta intencional à heterogeneidade dos grupos de crianças que frequentam atualmente as instituições. Assim, de modo a atingir os objetivos propostos, planeei algumas sugestões de interação que permitiam a utilização de estratégias diversificadas e a observação das crianças durante a utilização das mesmas. A metodologia utilizada contemplou uma pesquisa bibliográfica com base em vários referenciais teóricos e a recolha e análise de dados. Esta investigação possibilitou constatar que a Diferenciação Pedagógica subjacente às propostas apresentadas proporciona o desenvolvimento das crianças tanto a nível social como cognitivo; ABSTRACT: The present report reflects the research develop in the context of the Master Degree in Preschool Education and Teaching of the Primary School at University of Évora, and aims to obtain the degree of master in the levels mention above. This research was held in two different contexts, the first one in a pre-school classroom, and later in a classroom of fourth year of Primary School. The research performed aimed mainly to help me to understand what conditions must be gathered in order to respond appropriately to children's needs and to see how it could improve my actions and intervention by promoting meaningful and quality learnings, stressing that the purpose was not to provide the same for all children, but to each one what they needed. My bet on Pedagogical Differentiation and a flexible management of the curriculum comes to the principle of the right of all to learning and as an intentional response to the heterogeneity of the groups of children who currently attend institutions. Thus, in order to achieve the proposed objectives, I planned some suggestions for interacting with groups of children’s which allowed me to use multiple strategies and to do observation of the individuals while they were being applied. The methodology included a literature search based on various theoretical frameworks and data collection and the respective analysis. This research led us to confirm that the proposals based on pedagogical differentiation provide children's development both socially and cognitive and it is essential to ensure it since the earliest years of school, in order to build a rich differentiated pedagogical action and to promote learning for all students in pre-school and 1st cycle contexts by using diverse strategies.

Actividad biológica de bacteriocinas sobre el enquistamiento de Entamoeba histolytica HM1-IMSS y características nanomecánicas de la superficie celular al microscopio de fuerza atómica

La amibiasis es una infección parasitaria causada por Entamoeba histolytica. Representa una de las tres primeras causas de muerte por parásitos a nivel mundial. En México representa un problema de salud pública por su frecuencia, morbilidad, mortalidad y fácil dispersión. Muchos individuos infectados son portadores asintomáticos, lo que representa un reservorio para la diseminación a otros sujetos. Los pacientes infectados por E. histolytica eliminan trofozoítos no infecciosos y quistes infecciosos en sus heces. Los trofozoítos no pueden sobrevivir en el ambiente externo ni ser transportados a través del estómago si son ingeridos. La contaminación de alimentos y agua es la principal fuente para la transmisión de los quistes. La droga de elección para el tratamiento de la amibiasis y sus múltiples manifestaciones clínicas es el metronidazol, sin embargo presenta efectos secundarios indeseables en el humano y además existen reportes que indican que algunas cepas de E. histolytica presentan resistencia a esta droga, aunado a otros reportes que muestran actividad mutagénica y carcinogénica. Por esta razón se buscan alternativas terapéuticas para el tratamiento de esta enfermedad que no presenten efectos secundarios indeseables. Microorganismos producen sustancias antagónicas, por ejemplo, un amplio espectro de antibióticos y productos del metabolismo como ácidos orgánicos, moléculas quelantes de hierro (sideróforos) y bacteriocinas. Las bacteriocinas poseen espectros particulares de inhibición sobre el crecimiento de bacterias y protozoarios pero en enquistamiento tiene pocas investigaciones. En este proyecto se determinó la actividad biológica de bacteriocinas sobre el enquistamiento de E. histolytica HM1-IMSS bajo condiciones axénicas in vitro y se determinaron los cambios morfológicos tales como rugosidad, altura, ancho, volumen y longitud a través del microscopio de fuerza atómica (MFA) bajo el modo semi-contacto (tapping). Los datos se sometieron a un Análisis de Varianza (ANOVA) para determinar la significancia de los resultados.

Evaluación del efecto citopático inducido por un adenovirus oncolítico en una línea celular murina utilizada como modelo de cáncer cervical

Los adenovirus oncolífticos ofrecen un tratamiento muy prometedor para combatir al cáncer. Estos virus se replican pobremente en células de ratón, por lo que para evaluar su eficacia para destruir tumores, usualmente se implantan células tumorales humanas en las cuales se replican los virus, pero con el inconveniente que se deben usar ratones inmunosuprimidos, para evitar que su sistema inmune elimine estas células por ser de diferente especie. Sin embargo, estos ratones no representan un modelo adecuado para evaluar la eficacia terapéutica de dichos adenovirus en ensayos preclfnicos, ya que carecen de mecanismos de defensa propios del sistema inmune que pudiesen coadyuvar en la protección contra el tumor y/o eliminación del adenovirus. Actualmente no existe un modelo de cáncer de cervix en ratones silvestres (inmunocompetentes) en los cuales los tumores se desarrollen a partir de celulas murinas y que los virus oncolfticos se repliquen eficientemente, al igual que en las células humanas, y se permita evaluar su efecto antitumoral. Se ha demostrado que los genes E6 y E7 del HPV-16 facilitan la replicación de los adenovirus. En este trabajo por primera vez se evalu6 1a capacidad de replicación de un adenovirus oncolftico en Ia lfrrea celular murina TC-1 Ia cual posee los genes E6 y E7 del HPV-16. Demostramos que el adenovirus oncolitico Adhz60 indujo en Ia lfnea TC-1 un efecto citopatico evidente, disminuye importantemente su viabilidad celular, indujo la expresión de proteínas virales tempranas y tardas, fue capaz de generar una progenie viral infectiva y de inducir apoptosis. Contribuciones y Conclusiones. Con nuestros resultados demostramos que la linea celular murina TC-1 es permisiva para la replican del virus oncolftico Adhz60. Por lo tanto, las células TC-1 pueden ser usadas como modelo tumoral para evaluar adenovirus oncoliticos en ratones inmunocompetentes.

Imunidade celular: caracterização das proteínas celulares que inibem a infecção viral

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

Concreto celular polimérico: influência na adição de resíduo de poliéster insaturado termofixo

This work addresses the production of lightweight concrete building elements, such as plates, prefabricated slabs for pre-molded and panels of fencing, presenting a singular concrete: the Lightweight Concrete, with special properties such low density and good strength, by means of the joint use of industrial waste of thermosetting unsaturated polyesters and biodegradable foaming agent, named Polymeric Lightweight Concrete. This study covered various features of the materials used in the composition of the Polymeric Lightweight Concrete, using a planning of factorial design 23, aiming at studying of the strength, production, dosage processes, characterization of mechanical properties and microstructural analysis of the transition zone between the light artificial aggregate and the matrix of cement. The results of the mechanical strength tests were analyzed using a computational statistics tool (Statistica software) to understand the behavior and obtain the ideal quantity of each material used in the formula of the Polymeric Lightweight Concrete. The definition of the ideal formula has the purpose of obtaining a material with the lowest possible dry density and resistance to compression in accordance with NBR 12.646/92 (≥ 2.5 MPa after 28 days). In the microstructural characterization by scanning electron microscopy it was observed an influence of the materials in the process of cement hydration, showing good interaction between the wrinkled face of the residue of unsaturated polyesters thermosetting and putty and, consequently, the final strength. The attaining of an ideal formula, given the Brazilian standards, the experimental results obtained in the characterization and comparison of these results with conventional materials, confirmed that the developed Polymeric Lightweight Concrete is suitable for the production of building elements that are advantageous for construction

Síntese, estudos fotofísicos e imageamento celular de peptóides-cumarínicos fluorescentes baseados em Reações de Ugi

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2016.

Using induced pluripotent stem cells to investigate the mechanistic link between Gaucher disease and Parkinson related synucleinopathies

Dissertação de Mestrado, Ciências Biomédicas, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015