Algorithm for the Parkinson’s disease behavioural models characterization using a biosensor

Dissertação para obtenção do Grau de Mestre em Engenharia Biomédica

MOLECULAR CHARACTERIZATION AND SEQUENCE PHYLOGENETIC ANALYSIS OF SURFACE ANTIGEN 3 (SAG3) GENE OF LOCAL INDIAN ISOLATES (CHENNAI AND IZATNAGAR) OF Toxoplasma gondii

Context and objective:The molecular characterization of local isolates of Toxoplasma gondii is considered significant so as to assess the homologous variations between the different loci of various strains of parasites.Design and setting:The present communication deals with the molecular cloning and sequence analysis of the 1158 bp entire open reading frame (ORF) of surface antigen 3 (SAG3) of two Indian T. gondii isolates (Chennai and Izatnagar) being maintained as cryostock at the IVRI.Method:The surface antigen 3 (SAG3) of two local Indian isolates were cloned and sequenced before being compared with the available published sequences.Results:The sequence comparison analysis revealed 99.9% homology with the standard published RH strain sequence of T. gondii. The strains were also compared with other established published sequences and found to be most related to the P-Br strain and CEP strain (both 99.3%), and least with PRU strain (98.4%). However, the two Indian isolates had 100% homology between them.Conclusion:Finally, it was concluded that the Indian isolates were closer to the RH strain than to the P-Br strain (Brazilian strain), the CEP strain and the PRU strains (USA), with respect to nucleotide homology. The two Indian isolates used in the present study are known to vary between themselves, as far as homologies related to other genes are concerned, but they were found to be 100% homologous as far as SAG3 locus is concerned. This could be attributed to the fact that this SAG3 might be a conserved locus and thereby, further detailed studies are thereby warranted to exploit the use of this particular molecule in diagnostics and immunoprophylactics. The findings are important from the point of view of molecular phylogeny.

Effects of severe protein restriction in levels of parasitemia and in mortality of mice accutely infected with Trypanosoma cruzi

Adult mice were submitted to different degrees of protein restriction for five weeks (4.75, 9.5,14.25 and 19% of protein in isocaloric diets with normal content of mineral and vitamins), being subsequently infected with two strains of Trypanosoma cruzi: 10(5) trypomastigotes of Y strain or 14(5) trypomastigotes of CL strain. The same diet was maintained for all animals and the infection wasfollowed up by evaluation of blood parasites, mortality and intensity of lesions in the heart and skeleton muscle. Only severe protein restriction (4.75%) induced decrease in resistance to the infection with both the Y and CL strains of T. cruzi, which resulted in higher parasitemia and mortality. The inflammatory lesions in heart and skeleton muscle were less extensive in groups with severe protein restriction despite the increased number of parasite in muscle cells. Depression of immune mechanisms could be responsiblefor the reduced resistance and reduced inflammatory reaction after T. cruzi infection in severely protein restricted animals.

Heavy metal contamination in seafood and consumer exposure in the Gulf Cooperation Council

Dissertação para obtenção do Grau de Mestre em Tecnologia e Segurança Alimentar

Participatory Surveillance and Mathematical Models in Epidemiologic Research: Successes and Challenges

Theoretical epidemiology aims to understand the dynamics of diseases in populations and communities. Biological and behavioral processes are abstracted into mathematical formulations which aim to reproduce epidemiological observations. In this thesis a new system for the self-reporting of syndromic data — Influenzanet — is introduced and assessed. The system is currently being extended to address greater challenges of monitoring the health and well-being of tropical communities.(...)

Primary cilia and the regulation of hedgehog signaling: link to cardiac development

RESUMO: As células eucarióticas evoluíram um sistema de sinalização complexo que lhes permite responder aos sinais extracelulares e intracelulares. Desta forma, as vias de sinalização são essenciais para a sobrevivência da célula e do organismo, uma vez que regulam processos fundamentais, tais como o desenvolvimento, o crescimento, a imunidade, e a homeostase dos tecidos. A via de transdução de sinal Hedgehog (Hh) envolve o receptor Patched1 (Ptch1), que tem um efeito inibidor sobre a proteína Smoothened (Smo) na ausência dos seus ligandos, as proteínas Sonic hedgehog (Shh). Estas proteínas são reguladores fundamentais do desenvolvimento embrionário, como ilustrado pelas malformações drásticas observadas em embriões humanos e de murganho com perturbações da transdução de sinal da via Hh e que incluem polidactilia, defeitos craniofaciais e malformações ósseas. Igualmente importantes são as consequências da ativação inapropriada da via de sinalização Hh na formação de tumores. Curiosamente, os componentes desta via localizam-se nos cílios primários. Além disso, demonstrou-se que esta localização é crucial para a sinalização através da via Hh. Na presença dos ligandos, Ptch1 é internalizado e destinado a degradação ou sequestrado num compartimento da célula de onde não pode desempenhar o seu papel inibitório. A proteína Arl13b é uma pequena GTPase pertencente à família Arf/Arl da superfamília Ras de pequenas GTPases e foi implicada no síndrome de Joubert, uma ciliopatia caracterizada por ataxia congénita cerebelar, hipotonia, atrso mental e cardiopatia congénita. Murganhos deficientes para Arl13b, chamado hennin (hnn) morrem morrem prematuramente ao dia 13,5 de gestação (E13,5) e exibem anomalias morfológicas nos cílios que levam à interrupção da sinalização Hh. Além disso, a Arl13b está diretamente envolvida na regulação da via Hh, controlando a localização de vários componentes desta via nos cílios primários. Neste trabalho, mostramos que a Arl13b se localiza em circular dorsal ruffles (CDRs), que são estruturas de actina envolvidas em macropinocitose e internalização de recetores, e que regula a sua formação. Além disso, aprofundámos o conhecimento do processo de ativação da via de sinalização Hh, mostrando que as CDRs sequestram seletivamente e internalizam o recetor Ptch1. As CDRs formam-se minutos após ativação da via por ligandos Shh ou pelo agonista de Smo SAG e continuam a ser formadas a partir daí, sugerindo uma indução contínua da reorganização do citoesqueleto de actina quando a via está ativada. Observámos ainda que a inibição da formação de CDRs através do silenciamento de WAVE1, uma proteína necessária para a formação destas estruturas, resulta na diminuição da ativação da via de sinalização Hh. Além disso, o bloqueio da macropinocitose, que se segue ao fecho das CDRs, através do silenciamento de uma proteína necessária para a cisão de macropinossomas, nomeadamente a proteína BARS, tem um efeito semelhante. Estes resultados sugerem que as CDRs e a macropinocitose são necessárias para a ativação da via de sinalização Hh e indicam que esta via de internalização controla os níveis de sinal Hh. Durante o desenvolvimento, as células proliferativas dependem do cílio primário para a transdução de várias vias de sinalização. A via Hh induz a diferenciação do músculo cardíaco. Por conseguinte, os murganhos deficientes na via de sinalização Hh exibem uma variedade de defeitos de lateralidade, incluindo alteração do looping do coração, como pode ser visto em murganhos deficientes para Arl13b. Por conseguinte, investigámos o papel da Arl13b no desenvolvimento do coração. Mostramos que a Arl13b é altamente expressa no coração de embriões de murganho e de murganhos adultos ao nível do mRNA e da proteína. Além disso, o perfil de distribuição da Arl13b no coração segue o dos cílios primários, que são essenciais para o desenvolvimento cardíaco. Corações de murganhos hnn no estadio E12,5 mostram um canal átrio-ventricular aberto, espessamento da camada compacta ventricular e aumento do índice mitótico no ventrículo esquerdo. Além disso, um atraso de 1 a 2 dias no desenvolvimento é observado em corações de murganhos hnn, quando comparados com controlos selvagens no estadio E13,5. Assim, estes resultados sugerem que a Arl13b é necessária para o desenvolvimento embrionário do coração e que defeitos cardíacos podem contribuir para a letalidade embrionária de murganhos hnn. Em suma, foi estabelecido um novo mecanismo para a regulação dos níveis de superfície do recetor Ptch1, que envolve a remodelação do citoesqueleto de actina e a formação de CDRs após a ativação da via de sinalização Hh. Este mecanismo permite um feedback negativo que evita a repressão excessiva da via através da remoção de Ptch1 da superfície da célula. Além disso, determinou-se que uma mutação de perda de função na Arl13b causa defeitos cardíacos durante o desenvolvimento, possivelmente relacionados com a associação dos defeitos em cílios primários e na sinalização Hh, existentes em murganhos deficientes para Arl13b. A via de sinalização Hh tem tido um papel central entre as vias de sinalização, uma vez que a sua regulação é crucial para o funcionamento apropriada da célula. Assim, a descoberta de um novo mecanismo de tráfego através de macropinocitose e CDRs que controla a ativação e repressão da via de sinalização Hh traz novas perspetivas de como esta via pode ser regulada e pode ainda conduzir à identificação de novos alvos e estratégias terapêuticas. --------------------ABSTRACT: Eukaryotic cells have evolved a complex signaling system that allows them to respond to extracellular and intracellular cues. Signaling pathways are essential for cell and organism survival, since they regulate fundamental processes such as development, growth, immunity, and tissue homeostasis. The Hedgehog (Hh) pathway of signal transduction involves the receptor Patched1 (Ptch1), which has an inhibitory effect on Smoothened (Smo) in the absence of its ligands, the Sonic hedgehog (Shh) proteins. These proteins are fundamental regulators of embryonic development, as illustrated by the dramatic malformations seen in human and mouse embryos with perturbed Hh signal transduction that include polydactyly, craniofacial defects and skeletal malformations. Equally important are the consequences of inappropriate activation of the Hh signaling response in tumor formation. Interestingly, the components of this pathway localize to primary cilia. Moreover, it has been shown that this localization is crucial for Hh signaling. However, in the presence of the ligands, Ptch1 is internalized and destined for degradation or sequestered in a cell compartment where it no longer can play its inhibitory role. ADP-ribosylation factor-like (Arl) 13b, a small GTPase belonging to Arf/Arl family of the Ras superfamily of small GTPases has been implicated in Joubert syndrome, a ciliopathy characterized by congenital cerebellar ataxia, hypotonia, intellectual disability and congenital heart disease. Arl13b-deficient mice, called hennin (hnn) die at embryonic day 13.5 (E13.5) and display morphological abnormalities in primary cilia that lead to the disruption of Hh signaling. Furthermore, Arl13b is directly involved in the regulation of Hh signaling by controlling the localization of several components of this pathway to primary cilia. Here, we show that Arl13b localizes to and regulates the formation of circular dorsal rufles (CDRs), which are actin-basedstructures known to be involved in macropinocytosis and receptor internalization. Additionally, we extended the knowledge of the Hh signaling activation process by showing that CDRs selectively sequester and internalize Ptch1 receptors. CDRs are formed minutes after Hh activation by Shh ligands or the Smo agonist SAG and keep being formed thereafter, suggesting a continuous induction of actin reorganization when the pathway is switched on. Importantly, we observed that disruption of CDRs by silencing WAVE1, a protein required for CDR formation, results in down-regulation of Hh signaling activation. Moreover, the blockade of macropinocytosis, which follows CDR closure, through silencing of a protein necessary for the fission of macropinosomes, namely BARS has a similar effect. These results suggest that CDRs and macropinocytosis are necessary for activation of Hh signaling and indicate that this pathway of internalization controls Hh signal levels. During development, proliferating cells rely on the primary cilium for the transduction of several signaling pathways. Hh induces the differentiation of cardiac muscle. Accordingly, Hh-deficient mice display a variety of laterality defects, including alteration of heart looping, as seen in Arl13b-deficient mice. Therefore, we investigated the role of Arl13b in heart development. We show that Arl13b is highly expressed in the heart of both embryonic and adult mice at mRNA and protein levels. Also, Arl13b localization profile mimics that of primary cilia, which have been shown to be essential to early heart development. E12.5 hnn hearts show an open atrioventricular channel, increased thickening of the ventricular compact layer and increased mitotic index in the left ventricle. Moreover, a delay of 1 to 2 days in development is observed in hnn hearts, when compared to wild-type controls at E13.5. Hence, these results suggest that Arl13b is necessary for embryonic heart development and that cardiac defects might contribute to the embryonic lethality of hnn mice. Altogether, we established a novel mechanism for the regulation of Ptch1 surface levels, involving cytoskeleton remodeling and CDR formation upon Hh signaling activation. This mechanism allows a negative feedback loop that prevents excessive repression of the pathway by removing Ptch1 from the cell surface. Additionally, we determined that the Arl13b loss-offunction mutation causes cardiac defects during development, possibly related to the associated ciliary and Hh signaling defects found in Arl13b-deficient mice. Hh signaling has taken a center stage among the signaling pathways since its regulation is crucial for the appropriate output and function of the cell. Hence, the finding of a novel trafficking mechanism through CDRs and macropinocytosis that controls Hh signaling activation and repression brings new insights to how this pathway can be regulated and can lead to the discovery of novel therapeutic targets and strategies.

Quadrature generators based on ring oscillators and shift registers

Quadrature oscillators are key elements in modern radio frequency (RF) transceivers and very useful nowadays in wireless communications, since they can provide: low quadrature error, low phase-noise, and wide tuning range (useful to cover several bands). RC oscillators can be fully integrated without the need of external components (external high Q-inductors), optimizing area, cost, and power consumption. The conventional structure of ring oscillator offers poor frequency stability and phasenoise, low quality factor (Q), and besides being vulnerable to process, voltage and temperature (PVT) variations, its performance degrades as the frequency of operation increases. This thesis is devoted to quadrature oscillators and presents a detailed comparative study of ring oscillator and shift register (SR) approaches. It is shown that in SRs both phase-noise and phase error are reduced, while ring oscillators have the advantage of occupying less area and less consumption due to the reduced number of components in the circuit. Thus, although ring oscillators are more suitable for biomedical applications, SRs are more appropriate for wireless applications, especially when specification requirements are more stringent and demanding. The first architecture studied consists in a simple CMOS ring oscillator employing an odd number of static single-ended inverters as delay cells. Subsequently, the quadrature 4-stage ring oscillator concept is shown and post-layout simulations are presented. The 3 and 4-phase single-frequency local oscillator (LO) generators employing SRs are presented, the latter with 50% and 25% duty-cycles. The circuits operate at 600 MHz and 900 MHz, and were designed in a 130 nm standard CMOS technology with a voltage supply of 1.2 V.

Safety climate and its relationship with furniture companies’ safety performance and workers’ risk acceptance

This study aims to analyse the relationship between safety climate and the level of risk acceptance, as well as its relationship with workplace safety performance. The sample includes 14 companies and 403 workers. The safety climate assessment was performed by the application of a Safety Climate in Wood Industries questionnaire and safety performance was assessed with a checklist. Judgements about risk acceptance were measured through questionnaires together with four other variables: trust, risk perception, benefit perception and emotion. Safety climate was found to be correlated with workgroup safety performance, and it also plays an important role in workers’ risk acceptance levels. Risk acceptance tends to be lower when safety climate scores of workgroups are high, and subsequently, their safety performance is better. These findings seem to be relevant, as they provide Occupational, Safety and Health practitioners with a better understanding of workers’ risk acceptance levels and of the differences among workgroups.

The mineralogy and chemistry of the German and Portuguese tiles used to face a historic building in the Amazon region and their natural susceptibility to tropical weathering

During the 19th century, the most prominent buildings of the city of Belém were faced entirely with tiles manufactured in Portugal and Germany, which now exhibit distinct degrees of degradation. The Pinho mansion is one of the most important of these buildings and was selected for the investigation of the action of the tropical Amazonian climate on the degradation of the tiles. To achieve this objective, the tiles were mapped for organic and inorganic degradation, and samples were collected for analysis. The minerals were determined by XRD, the chemical composition by classical wet methods and SEM/EDS, and the microorganisms under the microscope. The results show that the German and Portuguese tiles are quite different in their composition. While both ceramic bodies are composed of SiO2 and Al2O3, CaO was found only in the Portuguese tile. The low Na2O and K2O contents indicate the addition of materials to reduce the fusion temperature. SiO2 and PbO are the main constituents of the glaze, with CoO and FeO being added as pigment. The ceramic body of the German tiles is constituted of quartz, mullite, and cristobalite, in contrast with the Portuguese tiles, which are made of quartz, gehlenite, diopside, calcite, and feldspars. The glazes are XRD-amorphous. The chemical and mineralogical differences between the German and Portuguese tiles indicate that they were produced from different raw materials under distinct thermal processes. The most prominent weathering-related modifications are the thin layers (German tiles), oxidation stains, dark stains, the detachment of the tile (Portuguese tiles), loss of the glaze and powdering of the ceramic body (Portuguese tiles) through the establishment of Cyanophyta and Bacillariophyta.. The distinct degradation patterns of the tiles exposed to the tropical Amazon climate are a consequence of their distinct mineralogy and chemistry.

Control of bovine mastitis: old and recent therapeutic approaches

Mastitis is defined as the inflammatory response resulting of the infection of the udder tissue and it is reported in numerous species, namely in domestic dairy animals. This pathology is the most frequent disease of dairy cattle and can be potentially fatal. Mastitis is an economically important pathology associated with reduced milk production, changes in milk composition and quality, being considered one of the most costly to dairy industry. Therefore, the majority of research in the field has focused on control of bovine mastitis and many efforts are being made for the development of new and effective anti-mastitis drugs. Antibiotic treatment is an established component of mastitis control programs; however, the continuous search for new therapeutic alternatives, effective in the control and treatment of bovine mastitis, is urgent. This review will provide an overview of some conventional and emerging approaches in the management of bovine mastitis infections.

Reproducibility and Reliability of the Quality of Life Questionnaire in Patients With Atrial Fibrillation

Abstract Background: Studies have shown the impact of atrial fibrillation (AF) on the patients' quality of life. Specific questionnaires enable the evaluation of relevant events. We previously developed a questionnaire to assess the quality of life of patients with AF (AFQLQ version 1), which was reviewed in this study, and new domains were added. Objective: To demonstrate the reproducibility of the AFQLQ version 2 (AFQLQ v.2), which included the domains of fatigue, illness perception and well-being. Methods: We applied 160 questionnaires (AFQLQ v.2 and SF-36) to 40 patients, at baseline and 15 days after, to measure inter- and intraobserver reproducibility. The analysis of quality of life stability was determined by test-retest, applying the Bartko intraclass correlation coefficient (ICC). Internal consistency was assessed by Cronbach's alpha test. Results: The total score of the test-retest (n = 40) had an ICC of 0.98 in the AFQLQ v.2, and of 0.94 in the SF36. In assessing the intra- and interobserver reproducibility of the AFQLQ v.2, the ICC reliability was 0.98 and 0.97, respectively. The internal consistency had a Cronbach's alpha coefficient of 0.82, compatible with good agreement of the AFQLQ v.2. Conclusion: The AFQLQ v.2 performed better than its previous version. Similarly, the domains added contributed to make it more comprehensive and robust to assess the quality of life of patients with AF.