DNA nanoprobes for molecular detection

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe main objective of this thesis was to develop a simple and inexpensive method for nucleic acid detection in biological samples based on the colorimetric properties of gold nanoparticles. Gold nanoparticles were synthesized and functionalized with thiol-modified DNA oligonucleotides (Au-nanoprobes) able to recognize a target sequence of interest. These Au-nanoprobes were characterized and then used in a colorimetric method for DNA detection with a single base resolution based on a non-cross-linking approach - upon increasing ionic strength, Au-nanoprobes aggregate and the solution changes color from the original red to blue, due to the red-shift of the typical Surface Plasmon Resonance (SPR) band of gold nanoparticles; the presence of a complementary target to the probe sequence, prevents aggregation of the Au-nanoprobes and the solution remains red. The use of gold-silver alloy nanoparticles functionalized with thiol-modified DNA oligonucleotides (AuAg-nanoprobes) to further develop a multiplex non-cross-linking method was also assessed. To carry out the proof-of-concept for single base mutation/Single nucleotide polymorphism (SNP) detection and further develop this non-cross-linking method, three different targets were selected considering clinical samples availability and the commercial interests of STAB VIDA, Lda. Single point mutations associated to β-thalassemia were chosen for the proof-of-concept. Later, to further evaluate the versatility of the method, two other targets were chosen: an SNP within the Diabetes and Obesity Regulated (DOR) gene (i.e. DOR1C/G), associated with diabetes and obesity; and a tag SNP within the Cytochrome P450 2D6 (CYP2D6) gene (i.e. CYP1846G/A), associated to a non-functional CYP2D6, which is responsible for the metabolism of xenobiotics. Studies were also conducted to better understand the underlying mechanisms involved in the non-cross-linking detection, towards the optimization of the method. The use of Atomic Force Microscopy (AFM), fluorescent spectroscopy and electrophoretic mobility measurements (Ferguson analysis) allowed clarifying the nature of the forces involved in the differential colorimetric non-cross-linking aggregation and further optimize the Au-nanoprobe design to SNP/single point mutation discrimination at room temperature. It has been demonstrated that the non-cross-linking method can be used for detection of SNP/single point mutations at room temperature. The detection sensitivity of the non-cross-linking method using Au-nanoprobes was determined to be 73 and 75 nM for ssDNA/dsDNA amplicons and ssDNA oligonucleotide targets, respectively.Fundação para a Ciência e Tecnologia (SFRH/BDE/15544/2005); Fundação Luso-Americana para o Desenvolvimento; STAB VIDA, Lda., especially the CEO, Orfeu Flore

Afirmação da identidade étnico-racial em crianças quilombolas e não quilombolas

AFFIRMATION OF ETHNIC-RACIAL IDENTITY IN QUILOMBOLA AND NON-QUILOMBOLA CHILDREN AFIRMACIÓN DE IDENTIDAD ÉTNICO-RACIAL EN NIÑOS QUILOMBOLAS Y NO QUILOMBOLAS ResumoAnalisa-se em dois estudos a identidade étnico-racial de crianças brancas, negras quilombolas e não quilombolas. No estudo 1, participaram 179 crianças negras e brancas, 54% de sexo feminino, 31% com idades entre seis e sete anos e o restante com idades entre oito e dez anos. Os resultados indicaram que as crianças brancas apresentam uma maior identificação com a cor da sua pele que as negras e que as negras não quilombolas são as que menos se auto identificam como negras. O segundo estudo, realizado oito meses depois, adotou um delineamento quase-experimental para testar o efeito de um conto de fadas na afirmação da identidade étnica das crianças negras. Participaram 29 crianças cujas identidades haviam sido analisadas no primeiro estudo, 16 de sexo feminino, sendo 12 delas com idades entre seis e sete anos. As crianças ouviam um conto de fadas cujos protagonistas eram todos negros e, em seguida, participavam da mesma entrevista do Estudo 1. Os resultados indicaram que a afirmação identitária se tornou mais forte, o reconhecimento de pertencimento étnico cresceu de 50% para mais de 70% e o sentimento de gostar de pertencer ao grupo aumentou também de forma significativa. No geral, os resultados obtidos apontam a importância integrada de Políticas Públicas de valorização identitária e ações voltadas para a representação simbólica dos grupos nos espaços de poder sobre a identidade étnica das crianças.Palavras-chave: Afirmação da identidade, Identidade negra, Infância, Quilombola. AbstractIn two studies, the ethnic-racial identity of white, black quilombola and non-quilombola children is analyzed. In study 1, 179 black and white children participated, 54% female, 31% aged between six and seven years and the remainder aged between eight and ten years. The results indicated that white children have a greater identification with the color of their skin than black women and that non-quilombola black women are the ones that least identify themselves as black. The second study, carried out eight months later, adopted a quasi-experimental design to test the effect of a fairy tale on the affirmation of the ethnic identity of black children. Twenty-nine children participated, whose identities had been analyzed in the first study, 16 female, 12 of them aged between six and seven years. The children listened to a fairy tale whose protagonists were all black and then participated in the same interview as in Study 1. The results indicated that the identity statement became stronger, the recognition of ethnic belonging grew from 50% to over 70 % and the feeling of enjoying belonging to the group also increased significantly. In general, the results obtained point to the integrated importance of Public Policies for the valorization of identity and actions aimed at the symbolic representation of groups in spaces of power over the children's ethnic identity.Keywords: Affirmation of identity, Black identity, Childhood, Quilombola ResumenEn dos estudios, se analiza la identidad étnico-racial de los niños blancos, negros quilombolas y no quilombolas. En el estudio 1, participaron 179 niños blancos y negros, 54% mujeres, 31% de edades comprendidas entre seis y siete años y el resto entre ocho y diez años. Los resultados indicaron que los niños blancos tienen una mayor identificación con el color de su piel que las mujeres negras y que las mujeres negras no quilombolas son las que menos se identifican como negras. El segundo estudio, llevado a cabo ocho meses después, adoptó un diseño cuasiexperimental para evaluar el efecto de un cuento de hadas en la afirmación de la identidad étnica de los niños negros. Participaron veintinueve niños, cuyas identidades habían sido analizadas en el primer estudio, 16 mujeres, 12 de ellas de entre seis y siete años. Los niños escucharon un cuento de hadas cuyos protagonistas eran todos negros y luego participaron en la misma entrevista que en el Estudio 1. Los resultados indicaron que la declaración de identidad se hizo más fuerte, el reconocimiento de pertenencia étnica creció del 50% a más de 70 % y la sensación de disfrutar pertenecer al grupo también aumentó significativamente. En general, los resultados obtenidos apuntan a la importancia integrada de las Políticas Públicas para la valorización de la identidad y las acciones dirigidas a la representación simbólica de los grupos en espacios de poder sobre la identidad étnica de los niños.Palabras llave: afirmación de identidade, Identidad negra, Infancia, Quilombol

Preliminary assessment of COVID-19 implications for the water and sanitation sector in Latin America and the Caribbean

COVID-19 has had a severe impact on human health, as well as in social and economic terms, with implications for the management and governance of the water and sanitation sector. These implications are evident in Latin America and the Caribbean due to existing challenges the region faces in accessing water and sanitation services. In spite of significant advances, around 65 million people in the LAC region currently lack appropriate access to water and soap to wash their hands—one of the most basic measures to prevent the spread of disease. Furthermore, social and economic vulnerabilities have exacerbated the effects of the pandemic in the region, particularly among those living in poverty. The COVID-19 pandemic thus requires the mobilization of frameworks such as the human rights to water and sanitation, specifically considering the region’s realities. This paper provides a review of some of the challenges currently faced in the region and advances a series of recommendations for enhancing access to water, sanitation and hygiene. The importance of effective governance, management and communication strategies in the water provisioning sector is highlighted in the context of the pandemic, and the role of science and research for adequate decision making is emphasized

Uso da avaliação focada com sonografia para trauma abdominal fechado

O trauma abdominal fechado, muitas vezes causado por acidentes de carro ou quedas, pode resultar em lesões graves na região do abdômen. É extremamente importante fazer um diagnóstico rápido e, frequentemente, o protocolo Advanced Trauma Life Support (ATLS) é utilizado para isso. No entanto, nem todas as lesões podem ser identificadas apenas com o exame físico, tornando o uso da Avaliação Focada com Sonografia para Trauma (FAST) essencial. O FAST é um exame de ultrassonografia rápido, não invasivo que procura por líquido livre na cavidade abdominal e é um indicador importante de lesões que podem exigir cirurgia ou tratamento imediato. Sua especificidade possui valores próximos a 100%, entretanto, a sensibilidade é variável - entre 30% a 80% - , visto que depende de diversos fatores, volume de perda sanguínea e experiência do operador. Sendo assim, embora seja muito útil em pacientes instáveis, a tomografia computadorizada com contraste ainda é considerada o método padrão para avaliações mais detalhadas em pacientes estáveis

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

The microbial ecology of a Mediterranean chlorinated drinking water distribution systems in the city of Valencia (Spain)

Drinking water distribution systems host extensive microbiomes with diverse biofilm communities regardless of treatment, disinfection, or operational practices. In Mediterranean countries higher temperatures can accelerate reactions and microbial growth that may increase aesthetic water quality issues, particularly where material deposits can develop as a result of net zero flows within looped urban networks. This study investigated the use of flow and turbidity monitoring to hydraulically manage mobilisation of pipe wall biofilms and associated material from the Mediterranean city of Valencia (Spain). Pipe sections of different properties were subjected to controlled incremental flushing with monitoring and sample collection for physico-chemical and DNA analysis with Illumina sequencing of bacterial and fungal communities. A core microbial community was detected throughout the network with microorganisms like Pseudomonas, Aspergillus or Alternaria increasing during flushing, indicating greater abundance in underlying and more consolidated material layers. Bacterial and fungal communities were found to be highly correlated, with bacteria more diverse and dynamic during flushing whilst fungi were more dominant and less variable between sampling sites. Results highlight that water quality management can be achieved through hydraulic strategies yet understanding community dynamics, including the fungal component, will be key to maintaining safe and ultimately beneficial microbiomes in drinking water distribution systems

What matters most? Stakeholders’ perceptions of river water quality

The need to integrate stakeholders’ views into environmental policy is increasingly gaining attention because this offers the opportunity to design sustainable and synergistic environmental strategies. Understanding and integrating the views of resource users into policy design and implementation could help address the most important challenges, gain community support, enhance project ownership, and avoid policies being rejected by local people. As a result, research in environmental management has focussed on stakeholders’ perceptions of river water quality and how to integrate such views into policy. While existing studies offer insights into the different ways in which stakeholders evaluate river water quality and potential factors influencing judgements, they appear to be limited in a number of ways. First, most of these studies focus on developed countries and may have limited contextual relevance to the developing world. Moreover, past studies focus on segments of society such as farmers and mainly on wastewater for agriculture. These shortcomings may limit our understanding of the topic and our ability to design effective policies to address water quality problems. Drawing on survey data from the Wenchi municipality in Ghana, we examine public perceptions of what constitute important measures of river water quality as well as factors influencing such judgements. Results suggest that while variables such as taste, colour, smell and litter are important, the presence of faecal matter in and/or around the river was rated the most important measure of river water quality while depth of river was the least important. Results further suggest that education, age, number of years a person had lived in a community, depth of river and the presence of aquatic vegetation influence water quality judgements. The findings of this research provide insights into what policymakers and regulators need to consider when attempting to influence behaviours in relation to water resources. We note, however, that while public perceptions of river water quality could guide water management policies, scientific measurements of water quality must not be replaced with stakeholder perceptions. This is because aspects such as ecological integrity may not be important to segments of the public but are an important aspect of water management. This is reinforced in the present study as there seems to be a lack of concern among the participants regarding river depth – an important factor for habitat provision and pollution dilution

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time, and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space. While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes, vast areas of the tropics remain understudied. In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity, but it remains among the least known forests in America and is often underrepresented in biodiversity databases. To worsen this situation, human-induced modifications may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge, it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost