Effects of UV radiation exposure on DNA and DNA repair enzymes

Dissertação para obtenção do Grau de Mestre em BiotecnologiaDNA integrity in the cell is under constant threat from damaging agents of endogenous or exogenous origin, such as UV light, ionizing radiation and oxidative stress. Although the effects of these carcinogens on DNA have been extensively studied, very little is known about their effect on DNA repair enzymes. The aim of the present work was the study of the effect of UV radiation on E. coli Endonuclease III, a DNA glycosylase belonging to base excision repair system. This enzyme was homologously overexpressed and then purified with a Fe/protein ratio of 3.88 ± 0.63 (fully‐loaded form). Endonuclease III exposure to UV radiation for 45 min (19.77 kJ dose) may lead to the destruction of the Fe‐S bonds of the [4Fe‐4S] cluster or to the conversion of this center into a different redox state. Electrophoretic mobility shift assays with protein‐DNA complex showed that Endonuclease III binding to plasmid DNA promotes a retardation of the free supercoiled DNA band, indicative of Endonuclease III‐DNA complex(es) formation. These assays also showed that Endonuclease III is able to bind both linear and supercoiled plasmid DNA, although with higher affinity for the linear form. Electrophoretic mobility shift assays performed after 45 min of UV irradiation (19.77 kJ) revealed that although shift occurred, the complexes formed were unstable and dissociated during electrophoresis. Moreover, the presence of aggregates suggests the unfolding of some Endonuclease III molecules. After 6 h of UV irradiation (158.18 kJ) no complexes are formed, leading to the conclusion that Endonuclease III molecules were irreversibly damaged. The electrochemical studies were performed by cyclic and differential pulse voltammetry techniques, at room temperature and anaerobic conditions; Endonuclease III and Endonuclease IIIDNA complex were adsorbed on a bare pyrolytic graphite electrode. For the first time, the direct electrochemical response of Endonuclease III unbound to DNA was observed, with a quasi‐reversible redox couple displaying a midpoint potential of 178 ± 9 mV vs. NHE. Endonuclease III binding to plasmid DNA promotes a positive shift (19 mV vs. NHE) in the characteristic redox couple of Endo III. Protein‐DNA complex UV irradiation promotes a negative shift in its redox potential of 25 mV vs. NHE

The first detection of Leishmania major in naturally infected Sergentomyia minuta in Portugal

Phlebotomine sandflies of the genus Sergentomyia are widely distributed throughout the Old World. It has been suggested that Sergentomyia spp are involved in the transmission of Leishmania in India and Africa, whereas Phlebotomus spp are thought to be the sole vectors of Leishmania in the Old World. In this study, Leishmania major DNA was detected in one Sergentomyia minuta specimen that was collected in the southern region of Portugal. This study challenges the dogma that Leishmania is exclusively transmitted by species of the genus Phlebotomus in the Old World.EU/FEDER [PTDC/CVT/112371/2009]; EU [FP7-261504 EDENext]info:eu-repo/semantics/publishedVersio

Application of kDNA as a molecular marker to analyse Leishmania infantum diversity in Portugal.

Around the Mediterranean basin Leishmania infantum is an important parasite causing canine leishmaniasis and visceral and cutaneous clinical forms in both immunocompetent and immunocompromised humans. Efficient monitoring and evaluation of epidemiology with discriminatory molecular markers are required. We investigated the genetic diversity of L. infantum in Portugal by polymerase chain amplification and restriction fragment length polymorphism analysis of kinetoplastid DNA, as molecular marker. We analysed 120 Portuguese isolates of L. infantum plus 16 other non-Portuguese isolates (as a reference group) from humans, dogs and sand flies. The Portuguese population showed a high degree of polymorphism with a total of 13 profiles identified. The predominant profile was A, which was only detected in the Portuguese samples. The kinetoplastid DNA PCR-RFLP assay described here was suitable for use directly with biological samples and the profiles obtained were stable during long-term growth in vitro and in laboratory animals

Whole school inspection nas escolas europeias : desafios e contradições

Relatório de estágio de mestrado, Educação e Formação (Área de especialidade em Organização e Gestão da Educação e da Formação), Universidade de Lisboa, Instituto de Educação, 2016O presente relatório pretende espelhar o meu percurso no âmbito de um estágio curricular na Inspeção-Geral da Educação e Ciência com a duração de nove meses, evidenciando as experiências, as aprendizagens e as competências que desenvolvi no âmbito do Programa de Avaliação Externa das Escolas e do Programa de Atividades Internacionais. Inserido no quadro da investigação naturalista, a metodologia seguida tem como suporte a pesquisa arquivística, a observação não participante e a entrevista semiestruturada, recorrendo à respetiva análise de conteúdo para tratamento dos dados. O relatório divide-se em três capítulos, complementados por um conjunto de anexos. O primeiro capítulo diz respeito à Caracterização da Inspeção-Geral da Educação e Ciência, abordando a cultura e o clima organizacional. O segundo capítulo aborda o Projeto de Investigação desenvolvido, aqui o foco é perceber qual é o papel da Whole School Inspection (WSI) nas Escolas Europeias. Para dar resposta a esta questão foram concebidos e trabalhados três eixos de análise: a caracterização das Escolas Europeias, a caracterização da Whole School Inspection e a análise e comparação dos guias que harmonizam esta intervenção avaliativa, atentando ao primeiro e ao segundo ciclos WSI (2011–2015 e 2016–2020), através da perspetiva de diversos atores e da análise do Common Framework for WSI 2011 e do Common Framework for WSI 2015. O terceiro capítulo refere-se às Atividades desenvolvidas no estágio, onde é realizada a descrição das tarefas executadas aquando da permanência na Inspeção-Geral da Educação e Ciência, bem como é apresentada uma reflexão sobre as aprendizagens e competências desenvolvidas e proporcionadas por essas atividades. A Whole School Inspection é, neste trabalho, perspetivada como um dispositivo de prestação de contas que, simultaneamente, tem em vista a melhoria da qualidade do serviço educativo prestado nas Escolas Europeias.This report is intended to reflect my route in scope of a traineeship in the Inspeção-Geral da Educação e Ciência with a duration of nine months, highlighting the experiences, learning and skills I developed in the External Program Evaluation for Schools and the International Program Activities. Framed in the naturalistic research, the methodology followed is supported by archival research, non-participant and participant observation, and semi-structured interview, appealed to the respective content analysis for data processing. The report is divided into three chapters complemented by a set of attachments. The first chapter concerns the Characterization of the Inspeção-Geral da Educação e Ciência, approaching to the culture and organizational climate. The second chapter refers the developed Research Project, here the focus is to understand what is the role of the Whole School Inspection (WSI) in the European Schools. To answer this question were designed and worked on three themes of analysis: the characterization of the European Schools, the characterization of the Whole School Inspection and the analysis and comparison of the guides that harmonize this evaluative intervention, referring to the first and second WSI cycles (2011-2015 and 2016-2020), through the perspective of various actors and the analysis of the Common Framework for WSI 2011 and the Common Framework for WSI in 2015. The third chapter refers to the stage developed activities, which is carried out the description of the tasks performed while within the Inspection General of Education and Science, and is presented a reflection on the learning and skills developed and provided by these activities. The Whole School Inspection external evaluation is in this work viewed as an accountability mechanism but simultaneously aims at improving the quality of educational services provided in the European Schools

Antioxidant and antiproliferative activities of the essential oils from Thymbra capitata and Thymus Species grown in Portugal

The antioxidant and antiproliferative activities of the essential oils from Thymbra capitata and Thymus species grown in Portugal were evaluated. Thymbra and Thymus essential oils were grouped into two clusters: Cluster I in which carvacrol, thymol, p-cymene, alpha-terpineol, and gamma-terpinene dominated and Cluster II in which thymol and carvacrol were absent and the main constituent was linalool. The ability for scavenging ABTS(center dot+) and peroxyl free radicals as well as for preventing the growth of THP-1 leukemia cells was better in essential oils with the highest contents of thymol and carvacrol. These results show the importance of these two terpene-phenolic compounds as antioxidants and cytotoxic agents against THP-1 cells.Fundacao para a Ciencia e Tecnologia (FCT) [Pest-OE/EQB/LA0023/2011

Diversidade genética da população parasitária de Leishmania em Portugal

O presente estudo teve como principal objectivo avaliar a diversidade genética de uma população parasitária de Leishmania em isolados portugueses de hospedeiros humanos, caninos, vulpinos e do vector, aplicando dois marcadores moleculares: kDNA e microssatélites. No Capítulo 1 fez-se uma revisão bibliográfica sobre as leishmanioses incluindo a epidemiologia da infecção nos países da bacia mediterrânica nomeadamente Portugal. Deu-se especial relevo à epidemiologia molecular que nos últimos anos tem vindo a ser desenvolvida. No Capítulo 2 efectuou-se um inquérito de leishmaniose canina que abrangeu 374 cães provenientes da Região Metropolitana de Lisboa. Foi encontrada uma prevalência total de 19,2%, com a prevalência de 18,4% nos cães com dono e 21,6% nos cães sem dono ou vadios. Os resultados obtidos evidenciaram a importância dos cães vadios na transmissão do parasita e disseminação da doença. A partir dos 72 cães infectados, foram isolados 49 estirpes de Leishmania, tendo estas sido tipadas como L. infantum zimodeme MON-1. Estas estirpes, em conjunto com outras amostras isoladas a partir de humanos, vector e outros canídeos, foram utilizadas para avaliar a diversidade genética. No Capítulo 3 foram desenvolvidas sequências iniciadoras cinetoplastideais, MC1 e MC2, tendo-se estas revelado específicas e sensíveis para a identificação do complexo L. donovani isolados em cultura ou directamente a partir de amostras clínicas. Aplicou-se a metodologia de kDNA-PCR-RFLP na análise de 161 amostras de DNA, das quais 134 eram provenientes de isolados portugueses de L. infantum. Foram identificados 16 genótipos na totalidade das amostras, tendo 13 sido identificados nas amostras portuguesas. Observou-se a predominância do genótipo A, observado exclusivamente na população parasitária portuguesa. Em termos geográficos esta metodologia mostrou estar de acordo com a tipagem isoenzimática, e outros marcadores moleculares, individualizando as amostras provenientes de África num único genótipo. No entanto não se observou individualização ao nível das regiões de Portugal estudadas, sugerindo a existência de fluxo genético entre as diferentes áreas geográficas. No Capítulo 4 aplicou-se a análise de 13 loci de microssatélites, polimórficos para L. infantum, em 154 amostras, das quais 128 eram provenientes de diferentes regiões geográficas de Portugal e de diferentes hospedeiros e vector. Obteve-se um maior grau de polimorfismo com estes marcadores do que com o kDNA, identificando-se 85 genótipos. Observou-se uma maior diversidade molecular nas amostras provenientes do Algarve e Alto Douro e, relativamente ao hospedeiro, estes alvos moleculares mostraram ser muito mais polimórficos no hospedeiro humano que o canino, indo ao encontro dos resultados de tipagem isoenzimática que se conhecem até à actualidade. Foi individualizado um agrupamento de amostras não MON-1 e dentro deste, um sub-agrupamento das amostras de África Oriental (Etiópia e Sudão), como anteriormente sugerido por outros autores. No Capítulo 5 discutiram-se os resultados obtidos ermitindo verificar que a variabilidade dos parasitas Leishmania no nosso país é maior do que tem sido considerada até ao presente. Possibilitaram também o conhecimento de que há genótipos predominantes em Portugal e que a variabilidade genética no hospedeiro humano e no vector é superior à do reservatório doméstico e silvático.The main objective of the present study was to access genetic diversity of a Leishmania parasite population from human, canine, vulpine and vector portuguese isolates, by using two molecular markers: kDNA and microsatellites. In Chapter 1, we have performed a bibliographic revision concerning leishmaniasis including epidemiology of the disease in the mediterranean countries namely Portugal. A special emphasis was given to molecular epidemiology that as been developed in the last years. In Chapter 2, we have performed a canine survey on leishmaniasis which included 374 dogs from the Lisbon metropolitan region. An overall prevelance of 19,2%, with 18,4% in domestic dogs and 21,6% in stray dogs were found. The results highlight the importance of stray dogs in parasite’s transmission and disease dissemination. From the 72 dogs that were found infected, 49 strains were isolated and typed as L. infantum MON-1. These strains, along with other human, vector and canine samples, were used to evaluate genetic diversity. In Chapter 3 we have developed kinetoplastid primers, MC1 and MC2, being these primers specific and sensitive for L. donovani complex identification in isolates from cultures or directly from clinical samples. kDNA-PCR-RFLP was applied on the analysis of 161 DNA samples, 134 of them belonging to L. infantum Portuguese isolates. 16 genotypes were identified in the total sample and 13 genotypes were identified in the portuguese samples. A predominance of genotype A was observed, which was exclusively found in the Portuguese parasite population. In terms of geographic distribution, this methodology showed to be in agreement with isoenzyme typing and with other molecular markers, separating a unique genotype for the samples from Africa. However, individualization was not observed in the studied geographic regions of Portugal suggesting the existence of genetic flow between the different regions. In Chapter 4, 13 microsatellite loci, polymorphic for L. infantum, were studied in 154 samples, 128 of them belonging to the different geographic regions of Portugaland different host and vector. A high degree of polymorphism was obtained with these markers than with kDNA, having identified 85 genotypes. Greater molecular diversity in samples from Algarve and Alto Douro was observed, and relatively to the host, human samples seemed to be more polymorphic than canine, which is in accordance with results from isoenzymatic typing that are currently known. A cluster of samples that are not MON-1 were individualized and within this group, a sub-cluster of samples of East Africa (Ethiopia and Sudan) as previously suggested by other authors. In Chapter 5, we have discussed the results obtained with both markers, kDNA and microsatellites, and the molecular methodologies applied. The results from this study allowed us to conclude that Leishmania parasites in Portugal resently show higher variability than what has been observed in the past. This study also allows us to know that some genotypes are predominant in Portugal and human host and vector genetic variabilities are higher than in domestic and silvatic reservoirs

LA EDUCACIÓN COMO ESTIMULANTE PARA LAS ACTITUDES LINGÜÍSTICAS POSITIVAS HACIA LAS LENGUAS NATIVAS DE COLOMBIA

This paper presents an action-research study on Language Attitudes toward indigenous languages at a Colombian public university. The Study addresses the analysis of the emerging sociolinguistic attitudes of a group of 39 students concerning minority languages. This research settles a comparison between two groups of a single population sample. Data collection was based on a structured survey to the students and documentary analysis. Expected results might determine the impact of these ideologies and the difference between those who have had contact with these languages and those who haven’t. In conclusion, understanding our perception of Indigenous languages would help us better understand their position in our society and give a better dimension to the danger they are inEste artículo presenta un estudio de investigación - acción sobre las actitudes lingüísticas hacia las lenguas indígenas en una universidad pública colombiana. El estudio aborda el análisis de las actitudes sociolingüísticas emergentes de un grupo de 39 estudiantes hacia las lenguas minoritarias. Esta investigación establece una comparación entre dos grupos de una misma muestra poblacional. La recolección de datos se basa en una encuesta estructurada a los estudiantes y un análisis documental. Los resultados esperados determinarían el impacto de estas ideologías y la diferencia entre aquellos que han tenido contacto con estas lenguas y aquellos que no. En conclusión, entender nuestra percepción de las lenguas indígenas nos ayudaría a comprender mejor su posición en nuestra sociedad y a dimensionar el peligro que corren

Consideraciones para el aprendizaje autónomo en el entorno asíncrono: caso de implementación gradual de aula invertida en ingeniería

[ES] El presente artículo aborda la implementación gradual en educación superior del modelo de aula invertida para el caso de estudiantes de primer semestre de ingeniería. La transición de clase tradicional hacia aula invertida inició en el entorno síncrono, dentro del aula, con la integración del aprendizaje activo basado en problemas, reportando resultados favorables. Sin embargo, los resultados muestran una disminución en el rendimiento relacionado con un mayor uso de TIC en el entorno asíncrono. En consecuencia, para lograr el aprendizaje autónomo, se propone considerar en la etapa de diseño del aula invertida, el estilo de aprendizaje, la disponibilidad de recursos tecnológicos y las competencias digitales de los estudiantesCortes Aguilar, TA.; Estelles Miguel, S. (2019). Consideraciones para el aprendizaje autónomo en el entorno asíncrono: caso de implementación gradual de aula invertida en ingeniería. En INNODOCT/18. International Conference on Innovation, Documentation and Education. Editorial Universitat Politècnica de València. 367-377. https://doi.org/10.4995/INN2018.2018.8778OCS36737

Leyendas tradicionales del pacifico como estrategia pedagógica de enseñanza-aprendizaje para el área de lenguaje del grado quinto del Centro Educativo Brisas de Pirisito, municipio de Roberto Payan, Nariño.

Con el desarrollo de esta propuesta se busca que por medio de una estrategia lúdico pedagógica implementada en el área de lenguaje formar en valores ancestrales a los estudiantes del grado quinto del centro educativo brisas de Pirisito para lo cual se realizó una entrevista a estudiantes, padres de familia en la que se identificó el problema, esto llevo a crear una malla curricular en la que se trabajaron tres logros del DBA y realizaron actividades como lectura de leyendas, las cuales incidieron favorablemente en el aprendizaje de los niños, aportaron también en el fortalecimiento de sus relaciones interpersonales con sus compañeros, los incitaron a practicar más valores humanos. Es el caso de la leyenda de la tunda ya que esta se llevaba a los niños groseros desobedientes, el simple hecho de saber que se podía convertir en cualquier persona que amaran hizo que bajaran el nivel de indisciplina, y grosería con las personas de la comunidad. Se realizó lectura y escritura de estas lo que mejoro su expresión ya que debían leer de forma adecuada y adoptar posturas correctas de lectura también les mejoro la caligrafía porque transcribieron y crearon sus propios textos como actividad final se realizó un dramatizado esto dejo como resultado: el conocimiento de las leyendas ancestrales por parte de los estudiantes, los motivo a seguir explorando su cultura y darla a conocer para que esta no se pierda, genero también un cambio de actitud en estos adoptando comportamientos adecuados hacia el prójimo esto se concluye en la importancia que tiene el acompañamiento de los padres en el proceso de crecimiento, y formativo de los hijos puesto que estos son el modelo a seguir de ahí la importancia en inculcarles valores, enseñarles la cultura para que ellos la conozcan aprendan a quererla y su lazo con ella sea tan fuerte que los prototipos de moda o la tecnología no generen su falta de práctica.With the development of this proposal, it is sought that by means of a pedagogic strategy implemented in the language area, ancestral values will be formed to the fifth grade students of the Brisas de Pirisito educational center, for which an interview was conducted with students, parents of family in which the problem was identified, this led to the creation of a curricular malla in which three DBA achievements were worked on and activities such as reading legends, which favorably affected the children's learning, also contributed to the strengthening of the their interpersonal relationships with their peers encouraged them to practice more human values. It is the case of the legend of the thrashing as it took away the rude disobedient children, the simple fact of knowing that it could become anyone they loved made them lower the level of indiscipline, and rudeness with the people of the community. Reading and writing of these was done, which improved their expression since they had to read correctly and adopt correct reading postures. They also improved their calligraphy because they transcribed and created their own texts as a final activity. A dramatization was carried out as a result: knowledge of ancestral legends by students, the reasons to continue exploring their culture and make it known so that it is not lost, also generate a change of attitude in these adopting appropriate behavior towards others this is concluded in the importance that has the support of parents in the process of growth, and training of children since these are the model to follow hence the importance in inculcating values, teaching them the culture so that they know they learn to love her and their bond with her is so strong that fashion prototypes or technology do not generate their lack of practice

1,2,4-Trioxolane and 1,2,4,5-Tetraoxane endoperoxides against old-world Leishmania parasites: in vitro activity and mode of action

Leishmaniasis remains one of the ten Neglected Tropical Diseases with significant morbidity and mortality in humans. Current treatment of visceral leishmaniasis is difficult due to a lack of effective, non-toxic, and non-extensive medications. This study aimed to evaluate the selectivity of 12 synthetic endoperoxides (1,2,4-trioxolanes; 1,2,4,5-tetraoxanes) and uncover their biochemical effects on Leishmania parasites responsible for visceral leishmaniasis. The compounds were screened for in vitro activity against L. infantum and L. donovani and for cytotoxicity in two monocytic cell lines (J774A.1 and THP-1) using the methyl thiazol tetrazolium assay. Reactive oxygen species formation, apoptosis, and mitochondrial impairment were measured by flow cytometry. The compounds exhibited fair to moderate anti-proliferative activity against promastigotes of the 2 Leishmania species, with IC50 values ranging from 13.0 ± 1.7 µM to 793.0 ± 37.2 µM. Tetraoxanes LC132 and LC138 demonstrated good leishmanicidal activity on L. infantum amastigotes (IC50 13.2 ± 5.2 and 23.9 ± 2.7 µM) with low cytotoxicity in mammalian cells (SIs 22.1 and 118.6), indicating selectivity towards the parasite. Furthermore, LC138 was able to induce late apoptosis and dose-dependent oxidative stress without affecting mithocondria. Compounds LC132 and LC138 can be further explored as potential antileishmanial chemotypes.info:eu-repo/semantics/publishedVersio