Papel da carga viral celular na imunodeficiência HIV/SIDA : contributo do estudo da infecção pelo HIV-2

Tese de doutoramento, Ciências Biomédicas (Imunologia), Universidade de Lisboa, Faculdade de Medicina, 2011The acquired immunodeficiency syndrome (AIDS), first described in 1981, has caused more than 25 million deaths worldwide with an estimated 2 million deaths related to disease in 2008, representing one of the most important public health problems worldwide. AIDS is caused by infection with human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). HIV-1 infection is pandemic, with more than 33 million people estimated to be infected, whilst HIV-2 infection remains relatively contained to a few West African countries, with Portugal representing the only non-African country with a significant HIV-2 prevalence (3.2% of infection cases reported in 2009). HIV-2 infection is characterized by a slower progression to AIDS as compared to HIV-1 infection, with a limited impact on the survival of the majority of infected adults. The rate of CD4 T cell decline is much slower than in HIV-1 and the levels of circulating virus (viremia) are significantly lower irrespective of disease stage. The low levels of viremia in patients infected with HIV-2 suggest a lower replicative activity. However, despite the lower viremia in HIV-2, our results, as well as studies from other groups, showed that levels of proviral DNA in peripheral blood mononuclear cells are similar in both infections, suggesting that a similar number of cells are infected. The cell tropism of HIV-2 and HIV-1 is determined by the expression of chemokine receptors on target cells in addition to the expression of the CD4 molecule. CCR5 and CXCR4 are the major co-receptors that are used for HIV-1 entry, and their levels of expression were shown to be directly related to the susceptibility to infection and to the rate of disease progression in HIV-1 infected individuals. Several studies suggest that, although HIV-2 can use a larger number of co-receptors as compared to HIV-1 in vitro, the major co-receptors used in vivo are also CCR5 and CXCR4. It is possible that different expression levels of these co-receptors lead to different patterns of infection in T cells populations determining the reduced production of virus and disease progression that is observed in individuals infected with HIV-2. We compared the expression of CCR5 and CXCR4 in patients infected with HIV-2 and HIV-1 that had similar levels of CD4 T cell depletion and found that patients infected with HIV-2 had an increased frequency of cells expressing CCR5 in the CD4 memory-effector population as compared to seronegative subjects. We also found a significant correlation between the frequency of cells expressing CCR5 in total CD4 T cells and the level of CD4 T cell depletion in individuals infected with HIV-2, but not in the ones infected with HIV-1. The expression of CCR5 is induced by cell activation and is usually restricted to the memory-effector population. We found the existence of a direct correlation between the frequency of cells expressing CCR5 and the levels of immune activation in individuals infected with HIV-2. These results demonstrate an association between the expansion of cells expressing CCR5 and immune activation in HIV-2. The lower frequency of cells expressing CCR5 in HIV-1 infected individuals, as compared with HIV-2+ individuals may be related to a continuous depletion of these cells in the presence of the higher levels of viremia associated with HIV-1 infection. Regarding the frequency of cells that express CXCR4 within CD4 T cell population, we found that this frequency was similar in patients infected with HIV-2 or HIV-1 and HIV-negative individuals. We also investigated the possibility that the similar levels of proviral DNA in the presence of different levels of viremia could be due to differences in the target cells. The levels of proviral DNA were quantified in purified naive and memory CD4 T cells from patients infected with HIV-2 and found to be very low in naive CD4 T cells, in marked contrast to the levels found in memory CD4 T cells. These results are in agreement with what is observed in individuals infected with HIV-1, suggesting that the memory CD4 T cell population is the main target for HIV-2, and further supporting the notion that CCR5 is the main co-receptor used by HIV-2 for infection in vivo. These data suggest that the lower viremia observed in patients infected with HIV-2 is not related to co-receptor availability on target cell populations. The low to undetectable levels of viremia in the presence of significant levels of proviral DNA may be due to the regulation of HIV-2 at the transcriptional level. Therefore, we proceeded with the study of viral mRNA expression through the development of methodologies based on real-time RT-PCR for the quantification of unspliced (gag) and multiply spliced (tat) mRNA transcripts of HIV-2 and HIV-1. The optimization of these approaches allowed the development of assays with high sensitivity, specificity and reproducibility, which were used to investigate the transcriptional activity in cohorts of HIV-1 and HIV-2 infected patients. We found that HIV-2 infected patients expressed significantly lower levels of tat mRNA as compared to patients infected with HIV-1. However, gag mRNA expression was found to be similar in the two cohorts. Given that tat mRNA seems to be mainly expressed in recently infected cells, our data suggest that the rate of new infections at the cellular level is lower in individuals infected with HIV-2 as compared to their HIV-1 infected counterparts. On the other hand, the observation of similar levels of gag mRNA in the two infections suggest that there is significant ongoing viral transcription in individuals infected with HIV-2, despite the low levels of viremia that characterize this infection. We further investigated the role of viremia, as well as levels of proviral DNA and tat and gag viral transcripts on the chronic immune hyperactivation observed in HIV-2 and HIV-1 infections. We found significantly higher levels of CD4 and CD8 T cell activation in HIV-2 infected individuals with detectable viremia as compared with aviremic HIV-2+ individuals. These results suggest a contribution of the circulating virus, even at low levels, to the state of immune activation found in these individuals. Our results further revealed a direct association between the levels of gag mRNA and the activation of CD4 T cells, supporting the possibility that persistent viral replication contributes significantly to the elevated immune activation levels observed in patients infected with HIV-2 despite the reduced viremia. In addition, we found a direct association between the levels of tat mRNA and CD8 T cell activation, particularly in individuals infected with HIV-1, suggesting that the levels of tat, or the number of newly infected cells, may have a role in CD8 T cell activation. Next, we evaluated the impact of antiretroviral therapy on the levels of viral transcripts and proviral DNA levels in treated HIV-2 infected individuals. Studies in treated HIV-1 patients showed that antiretroviral therapy led to decreased levels of viral mRNA, as well as a progressive decrease in the levels of proviral DNA. However, there were no data regarding the effects of antiretroviral therapy on the levels of viral transcripts and proviral DNA in treated HIV-2 infected individuals. We found that these individuals featured significantly higher levels of tat mRNA than their untreated counterparts, suggesting that the regimens used were not effective in reducing the rate of infection of new cells. We also documented the presence of mutations in reverse transcriptase and protease in the majority of treated patients, which indirectly supports the existence of persistent viral replication despite antiretroviral therapy. We further noted that these patients showed elevated levels of CD4 and CD8 T cell activation that negatively correlated with the number of CD4 T cells in circulation, possibly contributing to the reduced CD4 T cell recovery observed in these patients. In conclusion, we provide evidence of significant levels of viral replication in patients infected with HIV-2, and of its relationship with the levels of immune activation in spite of the low levels of circulating virus. Our data reinforce the importance of monitoring levels of persistent viral replication, even at low levels and emphasize the need to pursue clinical trials and studies on the efficacy of antiretroviral therapy in HIV-2 infection.POCI 2010 e o FSE. Bolsa de Doutoramento da Fundação para a Ciência e a Tecnologia (referência: SFRH/BD/23863/2005

Analgesic interpatient variability of remifentanil assessed through pupillary dilation reflex

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Pupillary pain index correlates with postoperative pain scores in neurosurgical patients

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Rapid prototyping and tooling in the manufacture of mould elements for large parts

In the last two decades the advances in rapid prototyping and tooling (RPT) technologies made possible the use of materials alternative to steel in some mould components. They also have allowed novel fabrication strategies and new ways of thinking on the production of injection moulds for short series of plas-tics parts. These technologies induced a new generation of toolmaking techniques. In the scope of the devel-opment project Hybridmould 21 the pros and cons of some RPT technologies were studied to fabricate moulding blocks and inserts for hybrid injection moulds for large plastics parts. Essentially, the studies aimed at finding the best solution for quick production and compliance with specific product requirements, such as complex features, complex cooling layout and large size (around 1 m2) in short production series. This paper discusses the main advantages and disadvantages associated to a number of different RPT technologies for the manufacture of moulding element.Program QREN - ‘Concessão de Incentivos Financeiros no âmbito do Sistema de Incentivos à Investigação e Desenvolvimento Tecnológico’ (contract 2010/013307) – Project Hybridmould 2

Análise do impacto do relevo na ocupação do solo urbano em zonas de montanha – Aplicação à cidade da Guarda, Portugal

O relevo é um dos fatores que influenciam o crescimento urbano e a sua morfologia. Este elemento natural, dependendo da sua intensidade, pode condicionar o desenvolvimento urbano sustentável. A existência de barreiras topográficas obriga à fragmentação do tecido urbano e consequente dispersão. Apresenta-se ao longo deste artigo a metodologia desenvolvida com o objetivo de analisar a integração das classes de uso e ocupação do solo urbano na topografia urbana. A análise espacial desenvolveu-se com recurso a Sistemas de Informação Geográfica. Os resultados obtidos permitiram concluir acerca da integração dos vários tipos de uso e ocupação do solo urbano no declive local; avaliar a existência de barreiras topográficas naturais no interior de espaços urbanos construídos, constituindo elementos fraturantes do crescimento urbano; analisar a inclusão de usos e ocupações de solo artificializado em zonas de risco. Esta análise constitui uma ferramenta de apoio ao planeamento urbano, na identificação de zonas de risco

Psychopathy Checklist-Revised (PCL-R) factor structure in male perpetrators of intimate partner violence

Psychopathy Checklist-Revised (PCL-R) is the gold standard for assessing psychopathy. However, its factorial structure has been subject of debate, and different factor models have been proposed. Furthermore, research has not focused on the PCL-R factorial structure among intimate partner violence (IPV) perpetrators. We analyzed and compared the fit of nine alternative models of PCL-R among a sample of 242 IPV perpetrators. Results revealed PCL-R has adequate factor validity. The three-factor model without testlets provided the best fit to the current data. The three-factor model also showed good reliability, and the factors established differential associations to penal and personal variables thus revealing discriminant validity. In the current study, psychopathy was thus best conceptualized as a clinical construct comprising a particular affective functioning (e.g., shallow affect, lack of empathy), and a specific interpersonal (e.g., egocentricity, deception) and behavioral style (e.g., irresponsibility, impulsivity) among IPV perpetrators.Portuguese Foundation for Science and Technology and the Portuguese Ministry of Science, Technology and Higher Education through national funds and co-financed by FEDER through COMPETE2020 under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007653). The study was also supported by Grant SFRH/ BD/66110/200

A study on the convergence of observer-based kinetics estimators in stirred tank bioreactors

This paper is devoted to the tuning problem of the "observer-based kinetics estimator" in stirred tank bioreactors. This algorithm estimates the reaction kinetics from the on-line knowledge of the state variables (either from measurement or by means of state observer), when the yield coefficients are known. The relation between the dynamics of convergence and the tuning procedure is explored. The method proposed imposes a second-order dynamics to the convergence of the estimator. This approach will be shown to compare favourably with a pole placement based technique, in an application to a baker's yeast fed-batch fermentation.Junta Nacional de Investigação Científica e Tecnológica (JNICT) - contract numbers BIC/636/92, BD/224/90-IF, BD/1476/91-RM

Urban sprawl assessment model in the context of sustainable development

The disordered urban growth may prove catastrophic for the future of cities if preventive measures are not taken. The increase of built-up areas, the growing need for implementation of urban infrastructures, the resulting increase in soil sealing, the excessive consumption of natural resources, the elevated need for car use on daily travels, the increase in travel distances, etc., are examples of the negative effects caused by urban sprawl. In order to implement measures to halt or avoid the damage caused by urban sprawl on the environment, society and economy, it is necessary to know which areas to intervene. In this sense, a multicriteria analysis model is presented that combines sustainable development indicators, with the main objective of calculating an index of sustainable urban expansion. This model, developed according to the Analytical Hierarchy Process, comprises three levels of analysis. At the first level, the indicators of urban sustainable development are aggregated and associated withsix criteria. The weighted aggregate of composite indicators, which constitute the second level of analysis, allows evaluating different scenarios and studying the level of influence of urban sprawl on the diverse dimensions. In the last level of analysis, an index is calculated,resulting from the aggregation of all indicatorsconsidered along the hierarchical structure. Thus, the proposed model allows quantifying the level of sustainability of urban sprawl, according to the lines of sustainable development defined for this work, serving as a tool to support urban planning interventions

Analyses of DNA damage in Saccharomyces cerevisiae mutant strains affected in the cell integrity (PKC) signaling pathway

The PKC signaling pathway is known to be related to genome integrity. Inclusively, it has already been shown that this pathway transmits information to the nucleus in what concerns genotoxic stress. Although this has been demonstrated it has not been shown that in the absence of a functional PKC pathway cells accumulate DNA damage. This research intends to investigate DNA damage caused by genotoxic stresses, as the one caused by the DNA replication inhibitor drug hydroxyurea, UV radiation and a genotoxic propolis extract, on mutants affected in the PKC pathway. The objective is to test the sensitivity of mutants affected in the genes BCK1, MKK1, MKK2 and SLT2, all fundamental MAPKs of this pathway. The biological model chosen for these experiments is Saccharomyces cerevisiae. As expected the results show higher sensitivity of the mutant strains when exposed to the genotoxicants, when compared to the parental strain, suggesting that PKC-defective mutants have decreased ability to repair DNA damage.info:eu-repo/semantics/publishedVersio

An integrated project of entrepreneurship and innovation in engineering education

This paper presents a new line of project based learning in the School of Engineering of University of Minho: the Innovation and Entrepreneurship Integrated Project (IEIP). Four groups, each one composed of students from different engineering integrated master courses – Mechanical, Industrial Electronics and Computers, Polymer, Industrial Management – compete against each other in developing or improving commercial products manufactured by actual industries. There have been so far five editions of the IEIP, with five different companies and five diverse products, however, all these products included components that required knowledge from all the engineering courses involved. Only with the cooperation between the students of the various courses that compose each multidisciplinary team, the success is attainable. As each student has to deal with various engineering scopes, students’ technical skills are greatly enlarged and they acquire a multidisciplinary knowledge that was not possible in another way. Their soft skills like project management, teamwork, communication ability and personal development, which are valuable requisites for their future employers, are also improved. The participating industries also take advantage of the project: the groups competing against each other act as a multiskilled work force, actually making proposals capable of improving their products, their efficiency, and reducing costs