Comparison of different techniques for mouse transgenesis

RESUMO: O uso de ratinhos transgénicos em neurociências aumentou consideravelmente nos últimos anos devido ao crescente interesse em compreender o cérebro e a necessidade de solucionar situações clínicas do foro neurológico e psiquiátrico. Para esse efeito, diferentes métodos de produção de animais transgénicos têm sido testados. O objectivo desta tese foi comparar métodos de integração aleatória de um transgene no genoma de ratinhos em termos de eficiência, estabilidade da integração do transgene, número de animais e de horas de trabalho necessárias para cada método. Assim, foi comparado o método mais utilizado - microinjecção pronuclear (PNMI) - com duas outras técnicas cujo desempenho foi considerado promissor – a transferência génica através dos testículos por electroporação e transfecção por lentivírus in vivo. As três técnicas foram realizadas usando um gene repórter sob o controlo de um promotor constitutivo, e depois reproduzidas usando um gene de interesse de modo a permitir obtenção de um animal capaz de ser usado em experimentação laboratorial. O transgene de interesse utilizado codifica uma proteína de fusão correspondendo a uma variante da rodopsina (channelrhodopsin) fundida à proteína enhanced yellow fluorescente protein ((EYFP) resultando num produto designado ChR2-EYFP. Este animal transgénico apresentaria expressão deste canal iónico apenas em células dopaminergicas, o que, com manipulação optogenética, tornaria possivel a activação especifica deste grupo de neurónios e, simultaneamente, a observação do impacto desta manipulação no comportamento num animal em livre movimento. Estas ferramentas são importantes na investigação básica em neurociências pois ajudam a esclarecer o papel de grupos específicos de neurónios e compreender doenças como a doença de Parkinson ou a esquizofrenia onde a função de certos tipos de neurónios de encontra alterada. Quando comparados os três métodos realizados verifica-se que usando um gene repórter PMNI resulta em 31,3% de, a de animais transgénicos obtidos, a electroporação de testículos em 0% e a injecção de lentivírus em 0%. Quando usado o gene de interesse, os resultados obtidos são, respectivamente, 18,8%, 63,9% e 0%.--------------ABSTRACT: The use of transgenic mice is increasing in all fields of research, particularly in neuroscience, due to the widespread need of animal models to solve neurological and psychiatric medical conditions. Different methodologies have been tested in the last decades in order to produce such transgenic animals. The ultimate goal of this thesis is to compare different methods of random integration of a transgene in the genome of mice in terms of efficiency, stability of the transgene integration, number of animals required and the labour intensity of each technique. We compared the most used method – pronuclear microinjection (PNMI) – with two other promising techniques – Testis Mediated Gene Transfer (TMGT) by electroporation and in vivo lentiviral transfection. The three techniques were performed using a reporter gene – green fluorescent protein (GFP), whose transcription was driven by the constitutive cytomegalovirus (CMV) promoter. These three techniques were later reproduced using the tyrosine hydroxylase promoter (TH) and the neuronal manipulator, channelrhodopsin-2 fused to the enhanced yellow fluorescent reporter protein (ChR2-EYFP). The transgenic animal we sough to produce would express the light driven channel only in dopaminergic cells, making possible to specifically activate this group of neurons, while simultaneously observe the behaviour in a freely moving animal. This is a very important tool in basic neuroscience research since it helps to clarify the role of specific groups of neurons, map circuits in the brain, and consequently understand neurological diseases such as Parkinson’s disease or schizophrenia, where the function of certain types of neurons is affected. When comparing the three methods, it was verified that using a reporter gene PNMI resulted in 31.3% of transgenic mice obtained, testis electroporation in 0% and lentiviral injection in 0%. When using the gene of interest, the results obtained were, respectively, 18.8%, 63.9% and 0%.

Economies of scale and scope in the provision of diagnostic techniques and therapeutic services in Portuguese hospitals

This paper analyses the provision of auxiliary clinical services that are typically carried out within the hospital. We estimate a exible cost function for the three most important (cost- wise) diagnostic techniques and therapeutic services in Portuguese hospitals: Clinical Pathology, Medical Imaging and Physical Medicine and Rehabilitation. Our objective in carrying out this estimation is the evaluation of economies of scale and scope in the provision of these services. For all services, we nd evidence of ray economies of scale and some evidence of economies of scope. These results have important policy implications and can be related to the ongoing discussion of where and how should hospitals provide these services.

Integration of forest fire management with SDI: user requirements.

Dissertation submitted in partial fulfilment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Integration of sediment contamination with multi–biomarker responses in a novel bioindicator candidate (Sepia officinalis) for risk assessment in impacted estuaries

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

Integration of crowdsourced information with traditional crises and disaster management information using linked data

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

Investigating the use of dasymetric techniques for assessing employment containment in Melbourne, Australia

Dissertation submitted in partial fulfillment of the requirements for the Degree of Master of Science in Geospatial Technologies.

The integration of online advertising: The effects of source expertise and certainty level of the message on consumer involvement and persuasion in the context of user-generated advertising

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA – School of Business and Economics

Integration of Membrane Filtration and Photolysis Processes for Drinking Water Treatment

Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Chemical Engineering.

An assessment of the integration of the mental health component into the care of women affected by intimate partner violence in Peru

RESUMO: A violência contra as mulheres (VCM) é um problema de saúde pública e uma violação dos direitos humanos. Ele tem uma alta prevalência na América Latina e no Caribe; o Estudo da Violência Contra as Mulheres da Organização Mundial de Saúde (OMS) identificou que as mulheres peruanas sofrem o maior índice de violência. O Perú é signatário da CEDAW e da Convenção de Belém do Pará, com recomendações para resolver este tipo de discriminação e descrever o papel do setor da saúde. A lei peruana define a violência como um problema de saúde mental. Objectivos: As três orientações clínicas do Ministério da Saúde para avaliar a integração da componente de saúde mental no cuidado de mulheres afetadas pela VCM foram revistas. Método: A proteção da saúde mental foi avaliada nas orientações acima mencionadas. A lei peruana relevante para perceber o reconhecimento das consequências de VCM na saúde mental e os cuidados prestados neste contexto foram revistos. Usando esses padrões nacionais e internacionais, foi realizada uma análise de conteúdo dos guias peruanos para a atenção da violência para ver como eles se integram a saúde mental. Resultados: Estas orientações são muito extensas e não definem claramente a responsabilidade dos profissionais de saúde. Não incluem um exame de saúde mental na avaliação da vítima e são vagas na descrição das atividades a serem realizadas pelo prestador dos cuidados de saúde. As orientações recomendam uma triagem universal usando um instrumento com formato antiquado e pesado. Em contrapartida, as orientações da OMS não recomendam qualquer triagem. Conclusão: As várias orientações analisadas não fornecem a informação necessária para o profissional de saúde avaliar o envolvimento da saúde mental e, desnecessariamente, tratam as mulheres sobreviventes de VCM como doentes mentais. Recomenda-se que as orientações recentes da OMS (Responding to intimate partner violence and sexual violence against women: WHO clinical and policy guidelines, 2013) para os cuidados de VCM sejam usadas como um modelo para o desenvolvimento de um único dispositivo técnico que incorpora directrizes com base científica. legislação com base no género, saúde, guias, prevenção e mujeres 6 RESUMO (PORTUGUESE) A violência contra as mulheres (VCM) é um problema de saúde pública e uma violação dos direitos humanos. Ele tem uma alta prevalência na América Latina e no Caribe; o Estudo da Violência Contra as Mulheres da Organização Mundial de Saúde (OMS) identificou que as mulheres peruanas sofrem o maior índice de violência. O Perú é signatário da CEDAW e da Convenção de Belém do Pará, com recomendações para resolver este tipo de discriminação e descrever o papel do setor da saúde. A lei peruana define a violência como um problema de saúde mental. Objectivos: As três orientações clínicas do Ministério da Saúde para avaliar a integração da componente de saúde mental no cuidado de mulheres afetadas pela VCM foram revistas. Método: A proteção da saúde mental foi avaliada nas orientações acima mencionadas. A lei peruana relevante para perceber o reconhecimento das consequências de VCM na saúde mental e os cuidados prestados neste contexto foram revistos. Usando esses padrões nacionais e internacionais, foi realizada uma análise de conteúdo dos guias peruanos para a atenção da violência para ver como eles se integram a saúde mental. Resultados: Estas orientações são muito extensas e não definem claramente a responsabilidade dos profissionais de saúde. Não incluem um exame de saúde mental na avaliação da vítima e são vagas na descrição das atividades a serem realizadas pelo prestador dos cuidados de saúde. As orientações recomendam uma triagem universal usando um instrumento com formato antiquado e pesado. Em contrapartida, as orientações da OMS não recomendam qualquer triagem. Conclusão: As várias orientações analisadas não fornecem a informação necessária para o profissional de saúde avaliar o envolvimento da saúde mental e, desnecessariamente, tratam as mulheres sobreviventes de VCM como doentes mentais. Recomenda-se que as orientações recentes da OMS (Responding to intimate partner violence and sexual violence against women: WHO clinical and policy guidelines, 2013) para os cuidados de VCM sejam usadas como um modelo para o desenvolvimento de um único dispositivo técnico que incorpora directrizes com base científica.-----------------ABSTRACT: Violence against women (VAW) is a public health problem and a human rights violation. It is highly prevalent in Latin America and the Caribbean; the Multi-country Study on Violence against Women by the World Health Organization identified rural Peruvian women as suffering the highest rates of VAW. The country is party to CEDAW and Belen Do Para Conventions, which set forth recommendations to overcome this form of discrimination and describe the role of the health sector. Peruvian law defines violence as a mental health issue. Objective: The Ministry of Health’s three technical guidelines were reviewed to assess the integration of mental health into the care of women affected by violence Method: The protection of the woman’s mental health was ascertained in the conventions mentioned above. The recognition of the mental health consequences of VAW and the inclusion of its evaluation and care were assessed in pertinent Peruvian legislation. Using these international and national parameters, the three guidelines for the attention of violence were subject to content analysis to see whether they conform to the conventions and integrate mental health care. Outcome: These guidelines are too extensive and do not clearly define the responsibility of health workers. They do not include a mental health exam in the evaluation of the victim and are vague in the description of the actions to be carried out by the health care provider. Guidelines prescribe universal screening using an outdated instrument and moreover, WHO Guidelines do not recommend screening. Conclusion: These multiple guidelines do not provide useful guidance for health care providers, particularly for the assessment of mental health sequelae, and unnecessarily stigmatize survivors of violence as mentally ill. It is recommended that the World Health Organization’s document Responding to intimate partner violence and sexual violence against women: WHO clinical and policy guidelines (2013) be used as a blueprint for only one technical instrument that incorporates evidence -based national policy and guidelines.

Analysis of the interaction of polycyclic aromatic compounds in a model organism: integration of genotoxic and histopathological effects

Due to their toxicity, especially their carcinogenic potential, polycyclic aromatic hydrocarbons (PAHs) became priority pollutants in biomonitoring programmes and environmental policy, such as the European Water Framework Directive. The model substances tested in this study, namely benzo[b]fluoranthene (B[b]F), considered potentially carcinogenic to humans and an effector carcinogenic PAH to wildlife, and phenanthrene (Phe), deemed a non-carcinogenic PAH, are common PAHs in coastal waters, owning distinct properties reflected in different, albeit overlapping, mechanisms of toxicity. Still, as for similar PAHs, their interaction effects remain largely unknown. In order to study the genotoxic effects of caused by the interaction of carcinogenic and non-carcinogenic PAHs, and their relation to histopathological alterations, juvenile sea basses, Dicentrarchus labrax, a highly ecologically- and economically-relevant marine fish, were injected with different doses (5 and 10 μg.g-1 fish ww) of the two PAHs, isolated or in mixture, and incubated for 48 h. Individuals injected with B[b]F and the PAH mixture exhibited higher clastogenic/aneugenic effects and DNA strand breakage in blood cells, determined through the erythrocytic nuclear abnormalities (ENA) and Comet assays, respectively. Also, hepatic histopathological alterations were found in all animals, especially those injected with B[b]F and the PAH mixture, relating especially to inflammation. Still, Phe also exhibited genotoxic effects in sea bass, especially in higher doses, revealing a very significant acute effect that was accordant with the Microtox test performed undergone in parallel. Overall, sea bass was sensitive to B[b]F (a higher molecular weight PAH), likely due to efficient bioactivation of the pollutant (yielding genotoxic metabolites and reactive oxygen species), when compared to Phe, the latter revealing a more significant acute effect. The results indicate no significant additive effect between the substances, under the current experimental conditions. The present study highlights the importance of understanding PAH interactions in aquatic organisms, since they are usually present in the aquatic environment in complex mixtures.

Integration of a mobile autonomous robot in a surveillance multi-agent system

This dissertation aims to guarantee the integration of a mobile autonomous robot equipped with many sensors in a multi-agent distributed and georeferenced surveillance system. The integration of a mobile autonomous robot in this system leads to new features that will be available to clients of surveillance system may use. These features may be of two types: using the robot as an agent that will act in the environment or by using the robot as a mobile set of sensors. As an agent in the system, the robot can move to certain locations when alerts are received, in order to acknowledge the underlying events or take to action in order to assist in resolving this event. As a sensor platform in the system, it is possible to access information that is read from the sensors of the robot and access complementary measurements to the ones taken by other sensors in the multi-agent system. To integrate this mobile robot in an effective way it is necessary to extend the current multi-agent system architecture to make the connection between the two systems and to integrate the functionalities provided by the robot into the multi-agent system.

Barriers preventing the successful integration of mental health services into Primary Health Care in Nigeria : a mixed methods approach

RESUMO: A Nigéria tem uma população estimada em cerca de 170 milhões de pessoas. O número de profissionais de saúde mental é muito diminuto, contando apenas com 150 psiquiatras o que perfaz aproximadamente um rácio de psiquiatra: população de mais de 1:1 milhão de pessoas. O Plano Nacional de Saúde Mental de 1991 reconheceu esta insuficiência e recomendou a integração dos serviços de saúde mental nos cuidados de saúde primários (CSP). Depois de mais de duas décadas, essa política não foi ainda implementada. Este estudo teve como objetivos mapear a estrutura organizacional dos serviços de saúde mental da Nigéria, e explorar os desafios e barreiras que impedem a integração bem-sucedida dos serviços de saúde mental nos cuidados de saúde primários, isto segundo a perspectiva dos profissionais dos cuidados de saúde primários. Com este objetivo, desenvolveu-se um estudo exploratório sequencial e utilizou-se um modelo misto para a recolha de dados. A aplicação em simultâneo de abordagens qualitativas e quantitativas permitiram compreender os problemas relacionados com a integração dos serviços de saúde mental nos CSP na Nigéria. No estudo qualitativo inicial, foram realizadas entrevistas com listagens abertas a 30 profissionais dos CSP, seguidas de dois grupos focais com profissionais dos CSP de duas zonas governamentais do estado de Oyo de forma a obter uma visão global das perspectivas destes profissionais locais sobre os desafios e barreiras que impedem uma integração bem-sucedida dos serviços de saúde mental nos CSP. Subsequentemente, foram realizadas entrevistas com quatro pessoas-chave, especificamente coordenadores e especialistas em saúde mental. Os resultados do estudo qualitativo foram utilizados para desenvolver um questionário para análise quantitativa das opiniões de uma amostra maior e mais representativa dos profissionais dos CSP do Estado de Oyo, bem como de duas zonas governamentais locais do Estado de Osun. As barreiras mais comummente identificadas a partir deste estudo incluem o estigma e os preconceitos sobre a doença mental, a formação inadequada dos profissionais dos CPS sobre saúde mental, a perceção pela equipa dos CSP de baixa prioridade de ação do Governo, o medo da agressão e violência pela equipa dos CSP, bem como a falta de disponibilidade de fármacos. As recomendações para superar estes desafios incluem a melhoria sustentada dos esforços da advocacia à saúde mental que vise uma maior valorização e apoio governamental, a formação e treino organizados dos profissionais dos cuidados primários, a criação de redes de referência e de apoio com instituições terciárias adjacentes, e o engajamento da comunidade para melhorar o acesso aos serviços e à reabilitação, pelas pessoas com doença mental. Estes resultados fornecem indicações úteis sobre a perceção das barreiras para a integração bem sucedida dos serviços de saúde mental nos CSP, enquanto se recomenda uma abordagem holística e abrangente. Esta informação pode orientar as futuras tentativas de implementação da integração dos serviços de saúde mental nos cuidados primários na Nigéria.------------ABSTRACT: Nigeria has an estimated population of about 170 million people but the number of mental health professionals is very small, with about 150 psychiatrists. This roughly translates to a psychiatrist:population ratio of more than 1:1 million people. The National Mental Health Policy of 1991 recognized this deficiency and recommended the integration of mental health into primary health care (PHC) delivery system. After more than two decades, this policy has yet to be implemented. This study aimed to map out the organizational structure of the mental health systems in Nigeria, and to explore the challenges and barriers preventing the successful integration of mental health into primary health care, from the perspective of the primary health care workers. A mixed methods exploratory sequential study design was employed, which entails the use of sequential timing in the combined methods of data collection. A combination of qualitative and uantitative approaches in sequence, were utilized to understand the problems of mental health services integration into PHC in Nigeria. The initial qualitative phase utilized free listing interviews with 30 PHC workers, followed by two focus group discussions with primary care workers from two Local Government Areas (LGA) of Oyo State to gain useful insight into the local perspectives of PHC workers about the challenges and barriers preventing successful integration of mental health care services into PHC. Subsequently, 4 key informant interviews with PHC co-ordinators and mental health experts were carried out. The findings from the qualitative study were utilized to develop a quantitative study questionnaire to understand the opinions of a larger and more representative sample of PHC staff in two more LGAs of Oyo State, as well as 2 LGAs from Osun State. The common barriers identified from this study include stigma and misconceptions about mental illness, inadequate training of PHC staff about mental health, low government priority, fear of aggression and violence by the PHC staff, as well as non-availability of medications. Recommendations for overcoming these challenges include improved and sustained efforts at mental health advocacy to gain governmental attention and support, organized training and retraining for primary care staff, establishment of referral and supportive networks with neighbouring tertiary facilities and community engagement to improve service utilization and rehabilitation of mentally ill persons. These findings provide useful insight into the barriers to the successful integration of mental health into PHC, while recommending a holistic and comprehensive approach. This information can guide future attempts to implement the integration of mental health into primary care in Nigeria.

Integration of speech-processing technologies into Activobank's client interaction process

This dissertation analyzes the possibilities of utilizing speech-processing technologies to transform the user experience of ActivoBank’s customers while using remote banking solutions. The technologies are examined through different criteria to determine if they support the bank’s goals and strategy and whether they should be incorporated in the bank’s offering. These criteria include the alignment with ActivoBank’s values, the suitability of the technology providers, the benefits these technologies entail, potential risks, appeal to the customers and impact on customer satisfaction. The analysis suggests that ActivoBank might not be in a position to adopt these technologies at this point in time.

The integration of online advertising into social media

This study examined user-generated (UG) advertising in the context of social media networks. The focus was on how people, whether an expert in the area, a non-expert or a friend, influence the reader of the advertisement. Furthermore, the study analyzed how the certainty level of the UG advertisement influences the person viewing the ad. The study showed that for the friend source a high certainty message was more persuasive. However, regarding the certainty no significant results were found for the expert and non-expert. Further, the type of the source had a considerable impact on persuasion. Someone that we personally know (e.g., a friend) was rated most positive for all analyzes variables. This shows that with the rising usage of social media there are great opportunities for new effective advertising strategies that could include a new type of an endorser – friends.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.