Characterization of the DNA methylation patterns of chemosensitive and chemoresistant human cancer cells: Biological and clinical impact

[spa] La quimioresistencia es el principal limitante para el tratamiento del paciente oncológico. La comprensión de los mecanismos celulares que conducen a la quimioresistencia puede tener un impacto drástico en la utilización de los fármacos. Estos agentes destruyen las células sensibles por inducción de programas de muerte celular. La desregulación de genes implicados en estos mecanismos puede ser una manera de quimioresistencia. Genes supresores tumorales y de reparación del ADN son importantes en estos procesos. Mientras que la inactivación de los primeros lleva a la quimioresistencia, la inactivación de los segundos puede conducir a quimosensibilidad. Mecanismos como la epigenética puede ser responsables por la alteración de estos genes. Por ejemplo, la hipermetilación del ADN en los promotores puede causar el silenciamiento de genes, y/o la hipometilación global producir la activación de otros genes. Pretendemos determinar si los cambios del perfil de metilación del ADN son importantes en la adquisición de resistencia a la quimioterapia. 1º estudio: 68 pacientes con cáncer colorectal (CCR) metastásico recibieron tratamiento con dacarbazina. 3% alcanzó una respuesta parcial y 12% la estabilización de la enfermedad. El mejor resultado se asoció a la hipermetilación del gen MGMT. 2º estudio: La resistencia al oxaliplatino depende en parte de la hipermetilación del gen SRBC. Este hecho predice una supervivencia libre de progresión más corta en pacientes tratados con oxaliplatino para los que no estaba indicada la cirugía de las metástasis. 3º Estudio: Descubrimos que el gen UGCG tiene un papel en resistencia de tumores testiculares germinales al cisplatino (CDDP). Sugerimos un agente químico que podría ser utilizado re-sensibilizar este tipo de tumores refractarios al CDDP. 4º estudio: MGMT se presenta hipermetilado en los modelos de cáncer testicular sensibles al cisplatino. En pacientes la hipermetilación de este gen encontrase asociada a una mayor supervivencia global. Se demostró que la inactivación de esto gen en tumores resistentes podría re-sensibilizarlos al CDDP. El estado de metilación de los promotores génicos influye en la sensibilidad tumoral a diferentes agentes quimioterapéuticos. Ponemos en evidencia que la inhibición de MGMT y UGCG pueden ser opciones terapéuticas para la rescatar pacientes refractarios al CDDP.[eng] Although chemotherapeutic drugs are widely used in order to improve the cancer outcome, drug resistance remain the most unpredictable factor affecting chemotherapy and a major impediment to successful patient’s treatment. Understanding the cellular mechanisms leading to chemoresistance may dramatically impact on the way chemotherapeutic drugs are used. Then, it would allow selecting the most suitable personalized therapy. It has become increasingly clear that many chemotherapeutic agents kill susceptible cells through the induction of the physiological cell death program. Accordingly, deregulation of any gene involved in the activation or execution of the death processes may be a major mechanism of chemoresistance. Tumor suppressor and DNA repair genes were classified as important mediators of chemotherapeutic respons. While inactivation of tumor suppressor genes could lead to drug resistance, inactivation of DNA repair genes, drug metabolisms, and detoxification genes might lead to drug sensitivity. This can be due to different mechanisms like regional hypermethylation and/or global hypomethylation. The possibility that some genes conferring chemoresistance are reversibly switched on/off by DNA methylation is particularly important and may have relevant clinical implications. A very potent specific inhibitor of DNA methylation, 5-AZA, has been widely used as a demethylating agent in vitro, and is used clinically in the treatment of acute leukemia and myelodysplasia. The present Doctoral Thesis has been devoted to provide further knowledge about the cross-talk between genes promoters DNA methylation status and tumors chemosensitivity, on a more detailed understanding of the influence of its changes in resistance to dacarbazine and to platinum agents, such oxaliplatin and cisplatin. We also pretend to explore alternative therapies as an attempt for reverse tumors chemoresistance the main cause of patients dead. In order to address these goals, we studied two cancer models, colorectal and testicular germ cell tumors. 1º Study: 68 patients were enrolled in this study and treated with dacarbazine. Overall, 2% achieved partial response and 12% had stable disease. Better outcome was significantly associated with MGMT promoter hypermethylation. 2º Study: We found that oxaliplatin resistance in colon cancer cells depends on the DNA methylation-associated inactivation of SRBC gene. SRBC over-expression or depletion gives rise to sensitivity or resistance to oxaliplatin, respectively. SRBC epigenetic inactivation occurred in primary tumors from a discovery cohort of colorectal cancer patients, predicting shorter progression free survival, particularly in oxaliplatin­treated cases for which metastasis surgery was not indicated. 3º Study: We report the perpetuation of serially cisplatin-refractory orthotopic transplantable patient-derived nonseminomatous tumor grafts in mice, named orthoxenografts, as a system to investigate cisplatin refractoriness from a genetic perspective and for the preclinical development of novel targeted therapies based on overcoming cisplatin-resistance. Here we found UGCG implicated in cisplatin resistance. By quimical inactivation we were able to revert cisplatin resistance in ortoxenograt tumors. 4º Study: To explore if MGMT promoter methylation changes have a role in cisplatin chemoresistance, we study it methylation status in cisplatin sensitive and paired resistant human non-seminoma cancer cell lines, xenograft tumors and in clinical samples from metastasic patients treated with cisplatin-based chemotherapy. We found that cisplatin sensitive samples are related with MGMT promoter hypermethylation associated with its loss of expression. Clinically, the presence of MGMT promoter methylation is related with better overall survival in metastasic patients with testicular germ cell cancer. Inhibition of MGMT with O6-benzylguanine in vitro or in vivo increases the sensitivity to cisplatin and temozolomide, being this a possible chemotherapeutic approach to re-sensibilize human non-seminoma refractory tumors. We conclude that the methylation status of the gene promoters influences tumor sensitivity to different chemotherapeutic agents. We demonstrated that inhibition of MGMT and UGCG can be therapeutic ways for rescue patient’s refractory to cisplatin

Psicose Orgânica em Doente com Tumor Carcinoide Atípico do Pulmão

Psychiatric manifestations are present in at least one third of cancer patients. Notwithstanding, less than 50% are properly followed‐up. This article contributes to the scarce existing literature on psychotic symptoms in patients with neuroendocrine tumors ‐ a rare presentation of a rare disease. It emphasizes a possible association between carcinoid tumor of the lung and psychotic symptoms, discusses the differential diagnosis and presents an effective psychopharmacological therapeutic option, never published before, to the best of our knowledge. We report the case of a female 69‐year‐old patient with an atypical carcinoid tumor of the lung and no previous psychiatric history. The clinical picture was characterized by paranoid and reference delusional ideas, with acoustic‐verbal hallucinatory activity, with secondary depressive mood and extreme anxiety. An organic psychosis was admitted with probable relationship with her neoplastic disease and was treated with aripiprazole, with good response and tolerance.Manifestações psiquiátricas estão presentes em pelo menos um terço dos doentes com cancro. Apesar disso, menos de 50% são devidamente acompanhados. Este artigo contribui para a escassa literatura existente sobre sintomas psicóticos em doentes com tumores neuroendócrinos ‐ uma apresentação rara de uma doença rara. Enfatiza uma possível associação entre tumor carcinoide do pulmão e sintomas psicóticos, discute o diagnóstico diferencial e apresenta uma opção terapêutica psicofarmacológica eficaz, nunca publicada, até onde sabemos. Relatamos o caso de uma doente de 69 anos com um tumor carcinoide atípico do pulmão, sem história psiquiátrica prévia. O quadro clínico caracterizou‐se por ideias delirantes paranoides e autoreferenciais, atividade alucinatória acústico‐verbal, humor depressivo e ansiedade extrema secundários. Foi admitida uma psicose orgânica em provável relação com a sua doença neoplásica, que foi tratada com aripiprazol, com boa resposta e tolerância

Prevalência de parasitas gastrointestinais em aves de falcoaria e psitacídeos no distrito de Lisboa

Orientação : Ana Munhoz ; co-orientação : Rui Filipe Galinho PatrícioAs aves podem ser acometidas por vários parasitas gastrointestinais tais como os helmintes e os protozoários. Estes têm vindo a destacar-se relativamente à frequência e aos numerosos problemas sanitários que provocam nas aves, selvagens e exóticas, mantidas em cativeiro. Vários estudos têm sido efetuados nos últimos anos, acerca da prevalência de parasitas gastrointestinais em aves de rapina e psitacídeos selvagens mas poucos têm sido efetuados nestas aves mantidas em cativeiro. Este trabalho pretende avaliar a prevalência dos parasitas gastrointestinais das aves de falcoaria e dos psitacídeos mantidos em cativeiro e a eventual necessidade de efetuar uma terapêutica preventiva e/ou curativa das parasitoses. Para tal foram realizados exames coprológicos através do método direto, método de flutuação e método de sedimentação em 121 aves, das quais 74 psitacídeos e 47 aves de falcoaria de varias espécies, idades e sexos, presentes em consultas na clínica vetolaias, oriundas de lojas, criadores e colecionadores. A presença de parasitas gastrointestinais foi observada em 4,1% dos psitacídeos e 14,9% nas aves de falcoaria. Este estudo demonstrou que, embora a prevalência observada não tenha sido tão elevada como descrita noutros estudos realizados, as aves em cativeiro também estão predispostas às parasitoses gastrointestinais, sendo aconselhável a adoção de medidas de prevenção.Birds can be affected by various gastrointestinal parasites such as protozoa and helminths. These have come to stand out in relation to the frequency and the many health problems that affect wild and exotic birds kept in captivity. Several studies have been made in recent years on the prevalence of gastrointestinal parasites in birds of prey and psittacine birds but few have been performade in these animals kept in captivity. This work intends to evaluate the situation of gastrointestinal parasites of falconry birds and psittacines in captivity and the possible need to make a preventive therapy and / or treatment of parasitic diseases. For this purpose stool tests were conducted through direct method, flotation method and sedimentation method in 121 birds, of which 74 were psittacines and 47 falconry birds of various species, ages and genders, present in consultations at the clinic vetolaias, coming from petshops, breeders and private collectors. The presence of gastrointestinal was observed parasites in 4.1% of the psittacines and 14.9% in the falconry birds. This study showed that, although the prevalence observed was not as high as described in other studies, the captive birds are also prone to gastrointestinal parasites, it is advisable to adopt preventive measures

a systematic review

The release of modified mosquitoes to suppress/replace vectors constitutes a promising tool for vector control and disease prevention. Evidence regarding these innovative modification techniques is scarce and disperse. This work conducted a systematic review, gathering and analysing research articles from PubMed and Biblioteca Virtual em Saúde databases whose results report efficacy and non-target effects of using modified insects for disease prevention, until 2016. More than 1500 publications were screened and 349 were analysed. Only 12/3.4% articles reported field-based evidence and 41/11.7% covered modification strategies’ post-release efficacy. Variability in the effective results (90/25.7%) questioned its reproducibility in different settings. We also found publications reporting reversal outcomes 38/10.9%, (e.g. post-release increase of vector population). Ecological effects were also reported, such as horizontal transfer events (54/15.5%), and worsening pathogenesis induced by natural wolbachia (10/2.9%). Present work revealed promising outcomes of modifying strategies. However, it also revealed a need for field-based evidence mainly regarding epidemiologic and long-term impact. It pointed out some eventual irreversible and important effects that must not be ignored when considering open-field releases, and that may constitute constraints to generate the missing field evidence. Present work constitutes a baseline of knowledge, offering also a methodological approach that may facilitate future updates.publishersversionpublishe

Leguminous cover crop to adapt olive rainfed orchards to climate change

Leguminous cover crops have the potential to contribute to soil protection against erosion and improve carbon sequestration, soil and water quality and might help agricultura! systems become more resilient to climate change. The aim of this study was to investigate the effects of a cover crop of self-reseeding annuallegumes of short growing cycle on physiological and biochemical responses of twenty-six-year-old olive trees (Olea europaea L. cv. Cobrançosa) grown under rainfed conditions, when compared with conventional tillage. The results showed that the leguminous cover crop ameliorates the physiological and biochemical performance of olive tree during the drought season, judging by the rise of net photosynthesis, transpiration rate and stomatal conductance, whereas the opposite pattern was observed for the concentration of total phenols and for total antioxidant activity in leaves. ln addition, it was observed an increase on yield, size and weight of olive fruits. These results indicate that the use of well-designed, locally-tailored cover crop mixtures, associated with an appropriate mowing time, is a very promising strategy to implement in olive orchards under water scarcity. Thus, cover cropping is a win-win strategy as it increases farm benefits and provides ecosystem services.This work was funded by the INTERACT project- "Jntcgrative Research in Environment, Agro-Chains and Technology", no. NORTE-01-0145-FEDER-000017, in its !ines of research entitled ISAC, co-tinanced by the European Regional Devclopment Fund (ERDF) through NORTE 2020 (North Regional Operational Program 20 14/2020). SM (PD/BD/135327/2017), EM (PD/BD/128274/2017) and CB (PD/BD/52543/2014) acknowledge the financ ial support provided by the FCT-Portuguese Foundatíon for Science and Technology, under the Doctoral Programme "Agricultura] Production Chains- from fork to farm" (PD/00122/2012).info:eu-repo/semantics/publishedVersio

Use of kaolin as a summer stress alleviating product in olive orchards under rainfed conditions

In a changing world, where is expected the temperatures rising and the reduction in precipitation in some semi-arid areas of the globe, the search for new agronomic practices that help crops to maintain and/or increase yields and quality is a continuous challenge. Kaolin (KL) exogenous application has been considered a short-term solution to alleviate the adverse effects of summer stress. The formed particle film increases the reflection of excess radiation reducing the risk of leaf damage from heat load accumulation and solar injury. Olive trees (Olea europaea L.) cultivated under rainfed conditions were sprayed with KL (5%) during two consecutive years in the beginning of the summer season. In general, relatively to the control group, KL-sprayed plants showed an enhancement in leaf water status, stomatal conductance and photosynthetic capacity and a reduction in leaf sclerophylly. In the severest period analyzed, at the end of the second-year summer, KL has lost the effectiveness of keeping gas exchange above the control measurements. However, in both years, allowed a faster restauration of the physiological functions in early autumn and contributed to higher crop yield. Overall, the results of the present investigation revealed that KL were effective in preventing the adverse effects of summer stress on crop performance and yield.Doctoral fellowship under the Doctoral Program “Agricultural Production Chains – from fork to farm” (PD/00122/2012) provided by the FCT-Portuguese Foundation for Science and Technology to C. Brito (PD/BD/52543/2014). Institution CITAB, for its financial support through the European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Program, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013.info:eu-repo/semantics/publishedVersio

A fertilização mineral com alta dose de azoto aumentou a produção de azeitona em olival de sequeiro, mas reduziu a matéria orgânica do solo em comparação com o uso de três corretivos orgânicos

Resíduos orgânicos de atividades domésticas ou agroindustriais devem ser reciclados, sendo o seu uso como corretivo de solo uma possibilidade interessante. Neste estudo, avaliouse durante quatro anos o efeito da aplicação de resíduos sólidos urbanos (RSU), estrume de curral (EC), cinzas de biomassa suplementadas com azoto (C+N), fertilização inorgânica comum na região (50 kg ha-1 de N, P20 S e K20) como testemunha (T) e T suplementada com 70 kg N ha- 1 (dose elevada de azoto, N+) em olival de sequeiro gerido com mobilização tradicional. O tratamento W aumentou a produtividade em comparação com os outros tratamentos (+ 165% do que RSU), tendo o N disponível mostrado ser a principal razão, em estreita associação com os incrementos da atividade fotossintética e da dimensão da superfície assimiladora a partir do terceiro ano do estudo. Por outro lado, em geral, as árvores tratadas com corretivos orgânicos apresentaram taxas fotossintéticas líquidas similares às plantas que receberam fertilização mineral convencional. As aplicações de RSU e EC aumentaram o teor de matéria orgânica no solo, os níveis de fósforo e a capacidade de troca catiónica, deixando boas indicações para o futuro, embora tenham fornecido pouco N às éÍrvores. O tratamento W reduziu significativamente o teor de matéria orgânica no solo (-63% do que RSU), resultado atribuído em parte ao sistema de gestão do solo por não permitir o desenvolvimento de vegetação herbácea, mas também a um efeito conhecido como interação do N adicionado, aspeto que compromete a sustentabilidade dessa estratégia de fertilização. Embora os RSU e as cinzas de biomassa possam estar associados a riscos de contaminação ambiental com metais pesados, neste estudo os níveis de metais no solo ou nos tecidos das plantas não foram preocupantes, pelo que a sua utilização nos olivais pode ser recomendada embora os seus efeitos devam continuar a ser monitorizados.Este trabalho foi financiado por Fundos Nacionais através da FCT - Fundação para a Ciência e a Tecnologia, no âmbito do projeto UIDB/04033/2020" e pelo Grupo Operacional Novas práticas em olivais de sequeiro: estratégias de mitigação e adaptação às alterações climáticas (Iniciativa ID 278).info:eu-repo/semantics/publishedVersio

Weather year-to-year variations determine the influence of kaolin and salicylic acid in olive fruits and oil phenolic composition

Olive tree (Olea europaea L.) growing area is threatened by the current and predicted adverse environmental conditions, affecting negatively the quality of the olive products, largely known for its benefits in human diet. Thus, agronomic strategies may be implemented to offset those negative effects. During two consecutive years young olive trees under rainfed conditions were sprayed in the beginning of the summer season with 100 M salicylic acid (SA), a stress signaling phytohormone, or with kaolin 5% (KL), a heat and irradiance reflecting clay. The distinct weather conditions during the fruit development stage, with higher rainfall close to the harvest and the early frost events, in the first year, and the warmer and drier summer, in the second year, contributed to the increase in phenolic compounds in fruits and in olive oil from the first to the second year. Both KL and SA increased crop yield, while, in general, phenolics concentrations of both olives and oil were increased and decreased by the applied products in the first and second year, respectively. This dissimilar response was modulated by the distinct weather conditions, demonstrating the effectiveness of the applied products in attenuating the frost-promoted degradation of phenolic compounds in the first year and in mitigating the extreme adverse conditions felt in the summer of the second year. The phenolic composition was also distinctly affected, since total phenols, ortho-diphenols and flavonoids exhibited different trends. It is hard to conclude a specific influence of the applied products on olives and oil phenolic composition, since the weather year-to-year variations determine different plant responses.Doctoral fellowship under the Doctoral Program “Agricultural Production Chains – from fork to farm” (PD/00122/2012) provided by the FCT-Portuguese Foundation for Science and Technology to C. Brito (PD/BD/ 52543/2014). Institution CITAB, for its financial support through the European Investment Funds by FEDER/COMPETE/POCI– Operational Competitiveness and Internationalization Program, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013.info:eu-repo/semantics/publishedVersio

Sustainable management of olive rainfed orchards by the introduction of leguminous cover crops

The olive sector has a crucial economic, social, cultural and ecological relevance in the Mediterranean region, where tillage and herbicides application are still generalized practices. However, these practices oppose the recommendations of UE Common Agricultural Policy and thus, management methods that decrease requirement for agricultural chemicals are needed to reduce adverse environmental impacts. Moreover, they can play an important role in atmospheric C01 emission and fixation. We propose green manure legumes to reduce erosion, soil compaction and nih·ate pollution, to improve biodiversity, soil carbon sink and fertility, to save fossil fuels and to increase yield and the safety and nutritional value of food products. The 3-yearfield study carried out in Northeast Portugal revealed that self-reseeding annual legumes is a promising strategy since increase the summer soil water content, the vegetative growth and the physiological tree pe1jormance, that was reflected in a 38% higher cumulative yield then tillage techniques.PTDC-AGR-AAM/098326/200