New molecularly-imprinted polymer for carnitine and its application as ionophore in potentiometric selective membranes

Carnitine (CRT) is a biological metabolite found in urine that contributes in assessingseveral disease conditions, including cancer. Novel quick screening procedures for CRT are therefore fundamental. This work proposes a novel potentiometric device where molecularly imprinted polymers (MIPs) were used as ionophores. The host-tailored sites were imprinted on a polymeric network assembled by radical polymerization of methacrylic acid (MAA) and trimethylpropane trimethacrylate (TRIM). Non-imprinted polymers (NIPs) were produced as control by removing the template from the reaction media. The selective membrane was prepared by dispersing MIP or NIP particles in plasticizer and poly(vinyl chloride), PVC, and casting this mixture over a solid contact support made of graphite. The composition of the selective membrane was investigated with regard to kind/amount of sensory material (MIP or NIP), and the need for a lipophilic additive. Overall, MIP sensors with additive exhibited the best performance, with near-Nernstian response down to ~ 1 × 10− 4 mol L− 1, at pH 5, and a detection limitof ~ 8 × 10− 5 mol L− 1. Suitable selectivity was found for all membranes, assessed by the matched potential method against some of the most common species in urine (urea, sodium, creatinine, sulfate, fructose and hemoglobin). CRT selective membranes including MIP materials were applied successfully to the potentiometric determination of CRT in urine samples.

Forest fire detection with a small fixed wing autonomous aerial vehicle

In this work a forest fire detection solution using small autonomous aerial vehicles is proposed. The FALCOS unmanned aerial vehicle developed for remote-monitoring purposes is described. This is a small size UAV with onboard vision processing and autonomous flight capabilities. A set of custom developed navigation sensors was developed for the vehicle. Fire detection is performed through the use of low cost digital cameras and near-infrared sensors. Test results for navigation and ignition detection in real scenario are presented.

Calibration Method for Underwater Visual Ground-Truth System

This work presents an automatic calibration method for a vision based external underwater ground-truth positioning system. These systems are a relevant tool in benchmarking and assessing the quality of research in underwater robotics applications. A stereo vision system can in suitable environments such as test tanks or in clear water conditions provide accurate position with low cost and flexible operation. In this work we present a two step extrinsic camera parameter calibration procedure in order to reduce the setup time and provide accurate results. The proposed method uses a planar homography decomposition in order to determine the relative camera poses and the determination of vanishing points of detected lines in the image to obtain the global pose of the stereo rig in the reference frame. This method was applied to our external vision based ground-truth at the INESC TEC/Robotics test tank. Results are presented in comparison with an precise calibration performed using points obtained from an accurate 3D LIDAR modelling of the environment.

Comparison of serology, antigenemia assay and the polymerase chain reaction for monitoring active cytomegalovirus infections in hematopoietic stem cell transplantation patients

Forty-six allogeneic hematopoietic stem cell transplantation (HSCT) patients were monitored for the presence of CMV antibodies, CMV-DNA and CMV antigens after transplantation. Immunoenzymatic serological tests were used to detect IgM and the increase in CMV IgG antibodies (increase IgG), a nested polymerase chain reaction (N-PCR) was used to detect CMV-DNA, and an antigenemia assay (AGM) was used to detect CMV antigens. The presence of CMV-IgM and/or CMV-increase IgG antibodies was detected in 12/46 (26.1%) patients, with a median time between HSCT and the detection of positive serology of 81.5 days. A positive AGM was detected in 24/46 (52.2%) patients, with a median time between HSCT and antigen detection of 62 days. Two or more consecutive positive N-PCR results were detected in 32/46 (69.5%) patients, with a median time between HSCT and the first positive PCR of 50.5 days. These results confirmed that AGM and mainly PCR are superior to serology for the early diagnosis of CMV infection. Six patients had CMV-IgM and/or CMV-increase IgG with a negative AGM (five cases) or N-PCR assay (one case). In five of these cases the serological markers were detected during the first 100 days after HSCT, the period of highest risk. These findings support the idea that serology may be useful for monitoring CMV infections in HSCT patients, especially when PCR is unavailable.

Molecularly imprinted sensor for voltammetric detection of norfloxacin

In this work, a norfloxacin selective modified glassy carbon electrode (GCE) based on a molecularly imprinted polymer (MIP) as electrochemical sensor was developed. A suspension of multi-walled carbon nanotubes (MWCNTs) was deposited on the electrode surface. Subsequently, a molecularly imprinted film was prepared by electropolymerization, via cyclic voltammetry of pyrrole (PPy) in the presence of norfloxacin (NFX) as the template molecule. A control electrode (NIP) was also prepared. Scanning electron microscopy (SEM) and cyclic voltammetry in a ferrocyanide solution were performed for morphological and electrochemical characterisation, respectively. Several experimental parameters were studied and optimised. For quantification purposes the MIP/MWCNT/GCE was immersed in NFX solutions for 10 min, and the detection was performed in voltammetric cell by square wave voltammetry. The proposed sensor presented a linear behaviour, between peak current intensity and logarithmic concentration of NFX between 1 × 10−7 and 8 × 10−6 M. The obtained results presented good precision, with a repeatability of 4.3% and reproducibility of 9% and the detection limit was 4.6 × 10−8 M (S/N = 3). The developed sensor displayed good selectivity and operational lifetime, is simple to fabricate and easy to operate and was successfully applied to the analysis of NFX in urine samples.

NEW PRIMERS FOR DETECTION OF Leishmania infantum USING POLYMERASE CHAIN REACTION

SUMMARY Leishmania infantum causes visceral leishmaniasis (VL) in the New World. The diagnosis of VL is confirmed by parasitological and serological tests, which are not always sensitive or specific. Our aim was to design new primers to perform a Polymerase Chain Reaction (PCR) for detecting L. infantum. Sequences of the minicircle kinetoplast DNA (kDNA) were obtained from GenBank, and the FLC2/RLC2 primers were designed. Samples of DNA from L. infantum, Leishmania amazonensis, Leishmania braziliensis, Leishmania guyanensis, Leishmania naiffi, Leishmania lainsoni, Leishmania panamensis, Leishmania major and Trypanosoma cruzi were used to standardize the PCR. PCR with FLC2/RLC2 primers amplified a fragment of 230 bp and the detection limit was 0.2 fg of L. infantum DNA. Of the parasite species assayed, only L. infantum DNA was amplified. After sequencing, the fragment was aligned to GenBank sequences, and showed (99%) homology with L. infantum. In the analysis of blood samples and lesion biopsy from a dog clinically suspected to have VL, the PCR detected DNA from L. infantum. In biopsy lesions from humans and dogs with cutaneous leishmaniasis, the PCR was negative. The PCR with FLC2/RLC2 primers showed high sensitivity and specificity, and constitutes a promising technique for the diagnosis of VL.

Gold nanoparticles for the detection of DNA adducts as biomarkers of exposure to acrylamide

The main objective of this thesis was the development of a gold nanoparticle-based methodology for detection of DNA adducts as biomarkers, to try and overcome existing drawbacks in currently employed techniques. For this objective to be achieved, the experimental work was divided in three components: sample preparation, method of detection and development of a model for exposure to acrylamide. Different techniques were employed and combined for de-complexation and purification of DNA samples (including ultrasonic energy, nuclease digestion and chromatography), resulting in a complete protocol for sample treatment, prior to detection. The detection of alkylated nucleotides using gold nanoparticles was performed by two distinct methodologies: mass spectrometry and colorimetric detection. In mass spectrometry, gold nanoparticles were employed for laser desorption/ionisation instead of the organic matrix. Identification of nucleotides was possible by fingerprint, however no specific mass signals were denoted when using gold nanoparticles to analyse biological samples. An alternate method using the colorimetric properties of gold nanoparticles was employed for detection. This method inspired in the non-cross-linking assay allowed the identification of glycidamide-guanine adducts and DNA adducts generated in vitro. For the development of a model of exposure, two different aquatic organisms were studies: a goldfish and a mussel. Organisms were exposed to waterborne acrylamide, after which mortality was recorded and effect concentrations were estimated. In goldfish, both genotoxicity and metabolic alterations were assessed and revealed dose-effect relationships of acrylamide. Histopathological alterations were verified primarily in pancreatic cells, but also in hepatocytes. Mussels showed higher effect concentrations than goldfish. Biomarkers of oxidative stress, biotransformation and neurotoxicity were analysed after prolonged exposure, showing mild oxidative stress in mussel cells, and induction of enzymes involved in detoxification of oxygen radicals. A qualitative histopathological screening revealed gonadotoxicity in female mussels, which may present some risk to population equilibrium.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection

Introduction Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. Methods We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. Results The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. Conclusions The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Influenza infection and Kawasaki disease

INTRODUCTION: The objective of this study was to investigate the possible link between influenza (Flu) infection and Kawasaki disease (KD). METHODS: We examined the medical records of 1,053 KD cases and 4,669 influenza infection cases hospitalized at our institute from January 1, 2011 to December 31, 2013. Cases of KD with concomitant influenza infection formed the KD + Flu group. Each KD + Flu case was matched with 2 KD cases and 2 influenza infection cases, and these cases were assigned to the KD group and Flu group, respectively. The differences in the principal clinical manifestations, course of disease, incomplete KD rate, intravenous immunoglobulin (IVIG) resistance rate, and echocardiographic detection results between the KD + Flu group and KD group were compared. The fever durations and laboratory test results of these three groups were compared. RESULTS: 1) The seasonal variations of the KD + Flu group, KD group and Flu group were similar. 2) The morbidity rate of incomplete KD was higher in the KD + Flu group compared with the KD group. 3) Patients in the KD + Flu group exhibited a longer time to KD diagnosis compared with patients in the KD group. 4) The KD + Flu group exhibited the longest fever duration among the three groups. 5) The CRP and ESR values in the KD + Flu group were higher those in the Flu or KD groups. CONCLUSIONS: Concomitant influenza infection affects the clinical manifestations of KD and can impact the laboratory test results and the diagnosis and treatment of the disease. However, it remains unclear whether influenza contributes to KD etiology.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Reinvigorating and redesigning early intervention in psychosis services for young people in Auckland

RESUMO: Auckland tem sido pioneira na implementação de modelos de Intervenção Precoce em Psicose. No entanto, esta organização do serviço não mudou nos últimos 19 anos. Segundo os dados obtidos da utilização do serviço, no período de 1996 -2012 foram atendidos 997 doentes, que tinham um número médio de 89 contactos (IQR: 36-184), com uma duração média de 62 horas de contactos (IQR: 24-136). Estes doentes passaram um número médio de 338 dias (IQR: 93-757) em contacto com o programa. 517 doentes (52%) não necessitaram de internamento no hospital, e os que foram internados, ficaram uma mediana de 124 dias no hospital (IQR: 40-380). Os doentes asiáticos tiveram um aumento de 50% de probabilidade de serem internados no hospital. Este relatório inclui 15 recomendações para orientar as reformas para o serviço e, nomeadamente, delinear a importância de uma visão organizacional e dos seus componentes-chave. As recomendações incluem o reforço da gestão e da liderança numa estrutura de equipe mais integrada, com recursos dedicados a melhorar a consciencialização da comunidade, a educação e deteção precoce, bem como a capacidade de receber referenciações diretas. Os Indicadores Chave de Desempenho devem ser estabelecidos, mas os Exames de Estado Mental em risco, devem ser removidos. Auckland deve manter a faixa etária alvo atual. A duração do serviço deve ser aumentada para um mínimo de três anos, com a opção de aumentá-la para cinco anos. A proporção de gestor de cuidados para os doentes deve ser preconizada em 1:15, enquanto o pessoal de apoio não-clínico deve ser aumentado. Os psiquiatras devem ter uma carga de trabalho de cerca de 80 doentes por equivalente de tempo completo. Um serviço local de prestação de cuidados deve ser desenvolvido com, nomeadamente, intervenções culturais para responder às necessidades da população multicultural de Auckland. A capacidade de investigação deve ser incorporada no Serviço de Intervenção Precoce em Psicoses. Qualquer alteração deverá envolver contacto com todas as partes interessadas, e a Administração Regional de Saúde deve comprometer-se em tempo, recursos humanos e políticos para apoiar e facilitar a mudança do sistema, investindo de forma significativa para melhor servir a comunidade Auckland.----------------------------------- ABSTRACT: Auckland has been pioneering in the adoption of Early Intervention in Psychosis models but the design of the service has not changed in 19 years. In service utilisation data from 997 patients seen from 1996 -2012, patients had a median number of 89 contacts (IQR: 36-184), with a median duration of 62 hours of contact (IQR: 24-136). Patients spent a median number of 338 days (IQR: 93-757) in contact with the program. 517 patients (52%) did not require admission to hospital, and those who did spent a median of 124 days in hospital (IQR: 40-380). Asian patients had a 50% increased chance of being admitted to hospital. This report includes 15 recommendations to guide reforms to the service, including outlining the importance of vision and key components. It recommends strengthened managerial leadership and a more integrated team structure with dedicated resources for improved community awareness, education and early detection as well as the capacity to take direct referrals. Key Performance Indicators (KPIs) should be established but At Risk Mental States should be excluded. Auckland should maintain the current target age range. The duration of service should be increased to a minimum of three years, with the option to extend this to five years. The ratio of care co-ordinator to patients should be capped at 1:15 whilst non-clinical supporting staff should be increased. Psychiatrists should have a caseload of about 80 per FTE. A local Service Delivery framework should be developed, as should cultural interventions to meet the needs of the multicultural population of Auckland. Research capacity should be incorporated into the fabric of Early Intervention in Psychosis Services. Any changes should involve consultation with all stakeholders, and the DHB should commit to investing time, human and political resources to support and facilitate meaningful system change to best serve the Auckland community.

Xenogeneic M2-polarization of Macrophages by undifferentiated and differentiated adipose tissue-derived stem cells

Mesenchymal stem cells (MSCs) are considered to be â â immunologically privileged.â â In a previous work when human adipose tissue-derived stem cells (hASCs) subcutaneously implanted in mice we did not identify an adverse host response1. Recently, it was shown that tissue regeneration could benefit from the polarization of M2 macrophages subpopulations 2. In this study we hypothesised that undifferentiated hASCs and derived osteoblasts and chondrocytes are able to switch murine bone marrow-derived macrophages (mBMMÃ s) into M2 phenotype, aiding tissue regeneration. Murine BMMÃ s were plated in direct contact with undifferentiated and osteo or chondro-differentiated hASCs for 4 h, 10 h, 24 h and 72 h. The cytokine profile was analysed by qRT-PCR and the surface markers were detected by flow cytometry. The direct interaction of both cell types was observed by time lapse microscopy. The results showed that mBMMÃ s polarized after contacting tissue culture polystyrene. This M2 phenotype was maintained along the experiment in direct contact with both undifferentiated and osteo or chondro-differentiated hASCs. This was confirmed by the expression of IL-1, IL-10, IL-4, TNF-a and IFN-g (genetic profile) and surface markers (CD206 + + , CD336 + + , MHC II + and CD86 + + ) detection. These data suggest the potential of hASCs in contemporary xenogenic tissue engineering and regenerative medicine strategies, as well as host immune system modulation in autoimmune diseases. 

Phenanthroimidazoles as fluorimetric and colourimetric chemosensors in aqueous solution

2,4,5-Triaryl-imidazoles are versatile compounds with application in medicine, due to their biological activity, and materials sciences, for their interesting optical properties. These properties can be tuned by careful selection of substituents at positions 2, 4 and 5: replacement of the aryl group by an heterocyclic group results in larger π-conjugated systems with improved optical properties for application in nonlinear optics, OLEDs, DNA intercalators, and chemosensors. Moreover, it is expected that introducing more conjugation and rigidity into the resulting system will further improve its properties. The development of chromo/fluorescent probes that are capable of detecting ions with high sensitivity and selectivity in aqueous media is currently a topic of strong interest and the design of heteroditopic receptors that contain two or more different binding sites for the simultaneous complexation of cationic and anionic guests is a emerging field of supramolecular chemistry. In this communication, we report the synthesis of new phenanthroimidazoles substituted at position 2 with arylthienyl or arylfuryl moieties possessing substituents of different electronic character, in order to tune the chromo/fluoro response in the presence of relevant anions and metal cations. Their photophysical properties and chemosensory ability were studied in acetonitrile and mixtures of acetonitrile and water, and selective detection of cyanide was achieved in aqueous mixtures for some of the derivatives.

Automatic visual detection of human behavior: a review from 2000 to 2014

Due to advances in information technology (e.g., digital video cameras, ubiquitous sensors), the automatic detection of human behaviors from video is a very recent research topic. In this paper, we perform a systematic and recent literature review on this topic, from 2000 to 2014, covering a selection of 193 papers that were searched from six major scientific publishers. The selected papers were classified into three main subjects: detection techniques, datasets and applications. The detection techniques were divided into four categories (initialization, tracking, pose estimation and recognition). The list of datasets includes eight examples (e.g., Hollywood action). Finally, several application areas were identified, including human detection, abnormal activity detection, action recognition, player modeling and pedestrian detection. Our analysis provides a road map to guide future research for designing automatic visual human behavior detection systems.

Insights into bacterial colony morphology evolution and diversification during infection development in cystic fibrosis

Tese de Doutoramento em Engenharia Biomédica.