Plasma versus serum analysis by FTIR spectroscopy to capture the human physiological state

Fourier Transform InfraRed spectroscopy of serum and plasma has been highly explored for medical diagnosis, due to its general simplicity, and high sensitivity and specificity. To evaluate the plasma and serum molecular fingerprint, as obtained by FTIR spectroscopy, to acquire the system metabolic state, serum and plasma spectra were compared to characterize the metabolic state of 30 human volunteers, between 90 days of consumption of green tea extract rich in Epigallocatechin-3-gallate (EGCG). Both plasma and serum spectra enabled the high impact of EGCG consumption on the biofluid spectra to be observed, as analyzed by the spectra principal component analysis, hierarchical-cluster analysis, and univariate data analysis. Plasma spectra resulted in the prediction of EGCG consumption with a slightly higher specificity, accuracy, and precision, also pointing to a higher number of significant spectral bands that were different between the 90 days period. Despite this, the lipid regions of the serum spectra were more affected by EGCG consumption than the corresponding plasma spectra. Therefore, in general, if no specific compound analysis is highlighted, plasma is in general the advised biofluid to capture by FTIR spectroscopy the general metabolic state. If the lipid content of the biofluid is relevant, serum spectra could present some advantages over plasma spectra.The present work was conducted at H&TRCHealth & Technology Research Center, Escola Superior de Tecnologia da Saúde de Lisboa, Instituto Politécnico de Lisboa, and in the Engineering and Health Laboratory, that resulted from a collaboration protocol established between Universidade Católica Portuguesa and Instituto Politécnico de Lisboa.info:eu-repo/semantics/publishedVersio

Comparação da funcionalidade de diferentes tipos de pés protésicos

Introdução - Amputação é uma cirurgia destrutiva / construtiva, nestas situações a restauração da capacidade da marcha só pode ocorrer mediante: uma adaptação na marcha, uma utilização ótima da musculatura remanescente e a utilização de uma prótese. Na bibliografia está descrito que 85% de todas as amputações são do membro inferior, sendo a amputação transtibial a amputação mais frequente; com uma predominância em indivíduos do sexo masculino (75% dos casos), com idades compreendidas entre os 50 e 75 anos e existindo um predomínio das amputações de etiologia vascular. De acordo com alguns autores, estes dados são também uma realidade em Portugal. Os indivíduos que sofrem uma amputação da extremidade inferior apresentam uma deterioração funcional variada, neste tipo de amputações, quer sejam amputados unilaterais ou bilaterais, transfemorais ou transtibiais, existem alterações na marcha que resultam numa diminuição da mobilidade. Existem diferentes fatores que podem influenciar o padrão de marcha de um amputado: derivados da protetização; que afetam a interface membro residual / prótese; relativos ao segmento intermédio da prótese; que dependem do mecanismo articular e os relacionados com a porção distal das próteses. Apesar destes fatores, os estudos em que se analisa o efeito do tipo de mecanismo protésico sobre a marcha do amputado transtibial são reduzidos. Não existindo bases científicas claras para a seleção dos mecanismos que melhor se adaptam a cada caso, usualmente o médico que prescreve a prótese escolhe uma ou outra, de acordo com as suas preferências pessoais, com a moda ou com a sua experiência profissional. Neste sentido torna-se importante desenvolver metodologias que possibilitem ao profissional prescritor avaliar, com dados quantificáveis qual a melhor escolha (personalizada ao paciente), quais os componentes mais indicados no fabrico de uma prótese. Objetivo do estudo - O objetivo deste estudo foi determinar qual dos pés protésicos melhor se adequa à funcionalidade do sujeito. Apresentando uma metodologia de avaliação de componentes protésicos (pés) que possa ser reproduzida periodicamente

Blood molecular profile to predict genotoxicity from exposure to antineoplastic drugs

This work was supported by Instituto Politécnico de Lisboa under grant IDI&CA/IPL/2021/PLASCOGEN_ESTeSL, IDI&CA/IPL/2017/GenTox/ESTeSL, and by the Fundação para a Ciência e a Tecnologia, Portugal, under grant DSAIPA/DS/0117/2020. The human biomonitoring had financial support given by the Portuguese Authority of Working Conditions (Project reference: 036APJ/09).Genotoxicity is important information that should be included in human biomonitoring programs. However, the usually applied cytogenetic assays are laborious and time-consuming, the reason why it is critical to developing rapid and economic new methods. The aim of this study was to evaluate if the molecular profile of frozen whole blood, acquired by Fourier Transform Infrared (FTIR) spectroscopy, allows to assess genotoxicity in occupational exposure to antineoplastic drugs, as obtained by the cytokinesis-block micronucleus assay. For that purpose, 92 samples of peripheral blood were studied: 46 samples from hospital professionals occupationally exposed to antineoplastic drugs and 46 samples from workers in academia without exposure (controls). It was first evaluated the metabolome from frozen whole blood by methanol precipitation of macromolecules as haemoglobin, followed by centrifugation. The metabolome molecular profile resulted in 3 ratios of spectral bands, significantly different between the exposed and non-exposed group (p<0.01), and a spectral principal component-linear discriminant analysis (PCA-LDA) model enabling to predict genotoxicity from exposure with 73 % accuracy. After optimization of the dilution degree and solution used, it was possible to obtain a higher number of significant ratios of spectral bands, i.e., 10 ratios significantly different (p<0.001), highlighting the high sensitivity and specificity of the method. Indeed, the PCA-LDA model, based on the molecular profile of whole blood, enabled to predict genotoxicity from exposure with an accuracy, sensitivity, and specificity of 92 %, 93 %, and 91 %, respectively. All these parameters were achieved based on 1 μL of frozen whole blood, in a high-throughput mode, i.e., based on the simultaneous analysis of 92 samples, in a simple and economic mode. In summary, it can be concluded that this method presents a very promising potential for high-dimension screening of exposure to genotoxic substances.info:eu-repo/semantics/publishedVersio

A simple, label-free, and high-throughput method to evaluate the epigallocatechin-3-gallate impact in plasma molecular profile

Epigallocatechin-3-gallate (EGCG), the major catechin present in green tea, presents diverse appealing biological activities, such as antioxidative, anti-inflammatory, antimicrobial, and antiviral activities, among others. The present work evaluated the impact in the molecular profile of human plasma from daily consumption of 225 mg of EGCG for 90 days. Plasma from peripheral blood was collected from 30 healthy human volunteers and analyzed by high-throughput Fourier transform infrared spectroscopy. To capture the biochemical information while minimizing the interference of physical phenomena, several combinations of spectra pre-processing methods were evaluated by principal component analysis. The pre-processing method that led to the best class separation, that is, between the plasma spectral data collected at the beginning and after the 90 days, was a combination of atmospheric correction with a second derivative spectra. A hierarchical cluster analysis of second derivative spectra also highlighted the fact that plasma acquired before EGCG consumption presented a distinct molecular profile after the 90 days of EGCG consumption. It was also possible by partial least squares regression discriminant analysis to correctly predict all unlabeled plasma samples (not used for model construction) at both timeframes. We observed that the similarity in composition among the plasma samples was higher in samples collected after EGCG consumption when compared with the samples taken prior to EGCG consumption. Diverse negative peaks of the normalized second derivative spectra, associated with lipid and protein regions, were significantly affected (p < 0.001) by EGCG consumption, according to the impact of EGCG consumption on the patients' blood, low density and high density lipoproteins ratio. In conclusion, a single bolus dose of 225 mg of EGCG, ingested throughout a period of 90 days, drastically affected plasma molecular composition in all participants, which raises awareness regarding prolonged human exposure to EGCG. Because the analysis was conducted in a high-throughput, label-free, and economic analysis, it could be applied to high-dimension molecular epidemiological studies to further promote the understanding of the effect of bio-compound consumption mode and frequency.info:eu-repo/semantics/publishedVersio

Evaluation of the Performance of Three Spacers

Agradecimentos às empresas GlaxoSmithKline (Portugal) e Astrazeneca (Suécia), pela cedência das câmaras de expansão Volumatic ® e NebuChamber®, respectivamente.Introdução: Existem vários factores que afectam a distribuição do fármaco nas vias áreas inferiores, onde podemos destacar os factores associados ao próprio dispositivo de inalação. Este trabalho tem como objectivo avaliar o desempenho da libertação de fármaco em três câmaras de expansão, assim como a quantidade de agente activo depositada no seu interior. Materiais e Métodos: Foi utilizado o Impinger Líquido de vários estágios (MSLI) de modo a avaliar o comportamento das partículas da suspensão Ventilan®HFA acoplado a três câmaras de expansão (Volumatic®, AeroChamber MAX® e NebuChamber®), de acordo com a Farmacopeia Portuguesa. A massa de sulfato de salbutamol recolhida nos diferentes compartimentos do impinger e no corpo da câmara foi determinada por espectrofotometria de modo a determinar a percentagem de massa cumulativa para cada câmara, determinando-se a fracção de partículas finas. Utilizou-se a Análise de Variância simples (One-way ANOVA) com o teste Post-Hoc de Bonferroni para a comparação dos resultados. Resultados: A deposição do sulfato de salbutamol ocorreu fundamentalmente no corpo das três câmaras, aproximadamente entre 40 a 50 %. Esta deposição é ligeiramente inferior na NebuChamber® (média±desvio-padrão: 43,8 % ± 11,6 %), mas não significativa relativamente à Volumatic® (p=0,351) ou à AeroChamber MAX® (p=0,115). A fracção de partículas finas assume valores de: 28,2 % ± 4,1 %, 29,6 % ± 2,4 % e 30,9 % ± 6,7 % para a Volumatic®, AeroChamber MAX® e NebuChamber®, respectivamente. Conclusão: As câmaras de expansão estudadas apresentam eficácias semelhantes na veiculação de sulfato de salbutamol aos estágios mais inferiores, não tendo sido encontrada relação entre os resultados e as suas características de volume, forma e material constituinte. Deste modo, a Volumatic® aparenta ser a câmara ideal para um hospital, uma vez que o seu grande volume não constitui uma desvantagem, sendo que o seu preço, inferior relativamente às anteriores, constitui uma vantagem de extrema importância para os hospitais públicos.Introduction: Several aspects are known to influence the drug distribution within the low respiratory tract, with particular emphasis on those related to the inhalation device. The aim of this work was to assess the performance of three spacers in the drug release, and also the quantity of active agent deposited inside these devices. Materials and Methods: In order to evaluate the behaviour of particles in suspension delivered through the Ventilan®HFA inhaler coupled to three different spacers (Volumatic®, AeroChamber MAX® and NebuChamber®) the Multistage Liquid Impinger (MSLI) was used, according to the Portuguese Pharmacopoeia. The mass of salbutamol sulphate deposited on the different impinger compartments and inside the spacer was determined by spectrophotometry, with the purpose of determining the percentage of cumulative mass for each spacer, and then the fine particle fraction. The results were compared statistically using a one-way analysis of variance (one-way ANOVA) with a Bonferroni post-hoc test. Results: About 40 to 50% of salbutamol sulphate was found deposited in the body of the three spacers. This deposition was slightly lower for NebuChamber® (average ± standard deviation of 43.8 % ± 11.6 %), in relation to Volumatic® (p=0.351) or AeroChamber MAX® (p=0.115). The fine particle fraction reached values of 28.2 ± 4.1%, 29.6 ± 2.4% and 30.9 ± 6.7% for Volumatic®, AeroChamber MAX® and NebuChamber®, respectively. Conclusion: The spacers showed to have similar efficiencies in the delivery of salbutamol sulphate in the last stages, and there was no relation between the results and the spacers characteristics such as volume, shape and material. Therefore, Volumatic® appears to be perfect for hospital use, since its big volume does not constitute a disadvantage, and its lower cost, when compared to the remaining two spacers, represents an advantage of utmost importance for public hospital

Evaluating the impact of culture conditions on human mesenchymal stem/stromal cell-derived exosomes through FTIR spectroscopy

In the last decade, the therapeutic effects of mesenchymal stem/stromal cells (MSCs) have been attributed to a paracrine activity exerted by extracellular vesicles secreted by MSCs, as exosomes. Their properties as intercellular communication vehicles have led to an increase interest in their use for cell-free therapeutic applications. The present work aimed to evaluate how different culture conditions, as culture medium (xenogeneic -free (XF) vs serum-containing medium), conditioning time (1, 2 and 3 days) and different MSC donors (n=6), affect the chemical characteristics of exosomes. For that, purified MSC-derived exosomes were characterized by Fourier-Transform InfraRed (FTIR) spectroscopy, a highly sensitive, fast and high throughput technique. The principal component analysis (PCA) of pre-processed FTIR spectra of purified exosomes was conducted, enabling the evaluation of the replica variance of the exosomes chemical fingerprint in a reduced dimensionality space. For that, different pre-processing methods were studied as baseline correction, standard normal variation and first and second derivative. It was observed that the chemical fingerprint of exosomes is more dependent of the medium used for MSCs cultivation than the MSC donor and conditioning days. Exosomes secreted by MSCs cultured with serum-containing medium presented a more homogenous chemical fingerprint than exosomes obtained with XF medium. Moreover, for a given medium (XF or serum-containing medium), the exosomes chemical fingerprint depends more of the MSC donor than of the conditioning days. The regression vector of the PCA enabled to identified relevant spectral bands that enabled the separation of samples in the score-plot of the previous analysis. Ratios between these spectral bands were determined, since these attenuate artifacts due to cell quantity and baseline distortions underneath each band. Statistically inference analysis of the ratios of spectral bands were conducted, by comparing the equality of the means of the populations using appropriate hypothesis tests and considering the significance level of 5%. It was possible to define ratios of spectral bands, that can be used as biomarkers, enabling the discrimination of exosomes chemical fingerprint in function of the medium used for MSC grown and the MSC donor. This work is therefore a step forward into understanding how different culture conditions and MSC donors affect MSC exosomes characteristics

New Tool for Signal Patients at Risk

Introduction: Pancreas transplantation is currently the only treatment that can re-establish normal endocrine pancreatic function. Despite all efforts, pancreas allograft survival and rejection remain major clinical problems. The purpose of this study was to identify features that could signal patients at risk of pancreas allograft rejection. Methods: We collected 74 features from 79 patients who underwent simultaneous pancreas–kidney transplantation (SPK) and used two widely-applicable classification methods, the Naive Bayesian Classifier and Support Vector Machine, to build predictive models. We used the area under the receiver operating characteristic curve and classification accuracy to evaluate the predictive performance via leave-one-out cross-validation. Results: Rejection events were identified in 13 SPK patients (17.8%). In feature selection approach, it was possible to identify 10 features, namely: previous treatment for diabetes mellitus with long-term Insulin (U/I/day), type of dialysis (peritoneal dialysis, hemodialysis, or pre-emptive), de novo DSA, vPRA_Pre-Transplant (%), donor blood glucose, pancreas donor risk index (pDRI), recipient height, dialysis time (days), warm ischemia (minutes), recipient of intensive care (days). The results showed that the Naive Bayes and Support Vector Machine classifiers prediction performed very well, with an AUROC and classification accuracy of 0.97 and 0.87, respectively, in the first model and 0.96 and 0.94 in the second model. Conclusion: Our results indicated that it is feasible to develop successful classifiers for the prediction of graft rejection. The Naive Bayesian generated nomogram can be used for rejection probability prediction, thus supporting clinical decision making.publishersversionpublishe

SIG e património: A experiência do inventário geo-referenciado do património metropolitano

Os Sistemas de Informação Geográfica (SIG) eram, tradicionalmente, uma metodologia de trabalho pouco utilizada em inventários de património. Nos últimos anos, diversas entidades empreenderam esforços para melhorar as suas bases de dados de património, enriquecendo-as com a possibilidade de, a par com a pesquisa alfanumérica, poder realizar também pesquisas e análises espaciais. Utilizando como ponto de partida dados provenientes do projecto “Corredores Verdes para a Área Metropolitana de Lisboa”, actualizando-os e aperfeiçoando-os, o inventário geo-referenciado do património da Área Metropolitana de Lisboa, criou – através da construção de um Sistema de Informação Geográfica – uma base de dados rigorosa e actualizável. Este SIG Património permite não só a pesquisa sobre o património classificado e não classificado da área metropolitana (num total de quase 4000 fichas, correspondendo a elementos e conjuntos geo-referenciados), mas também o cruzamento com outros tipos de informação (por exemplo, altimetria, hidrografia ou dados sobre a população). Nesta comunicação apresentamos o modo como foi elaborado este SIG Património, seguindo os passos dados para a sua construção, assim como os critérios utilizados e as dificuldades que surgiram durante todo o processo.info:eu-repo/semantics/publishedVersio

Public Art Journal

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Biomarkers research for diagnosis and prognosis of rejections of renal allograft in human transplant by fourier transform infrared spectroscopy

Mestrado em Engenharia BiomédicaABSTRACT - Background and main goals: Renal transplantation, when possible, is the treatment of choice for end-stage kidney disease as it enables a much higher quality of life than dialysis. However, one of its major problems is allograft rejection. The present project aims to promote serum biomarkers discovery for diagnosis and prognosis of the rejection processes of allograft organs and to evaluate the efficiency of organ rescue therapies, in a rapid, economic but also sensitive and specific mode. It was aimed to develop a new biomarker discovery methodology based on Fourier Transformed Infrared (FTIR) spectroscopy associated with multivariate data analysis and machine learning techniques. Methods: A total of 38 healthy non-transplanted participants, 59 transplanted patients with kidney allograft, from which 12 also received an allograft pancreas or liver, were considered. From the transplanted patients, 29 presented rejection processes. The FTIR spectra were acquired from serum samples of non-transplanted (n=38) and transplanted without (n=213) and with rejection (n=360) processes. It was optimized the dilution degree of the serum samples before spectra acquisition and the spectra pre-processing methods. Diverse spectra unsupervised and supervised processing multivariate data analysis were implemented to search for patterns in data and to develop classification methods to predict the diagnosis, prognosis, and immune mechanisms of rejection and the efficiency of the organ rescue treatment. It was also developed an in vitro method based on T-lymphocyte spectra to detect T-cells activation. This assay was based on whole blood samples of 8 healthy non-transplanted volunteers. Results: It was possible to develop good classification models to predict which patients will develop a rejection process, as for example by Random Forest an AUC of 0.94 was obtained. It was also possible to develop good models to predict the risk of the rejection process, as early as 120 days before it was detected in biopsies. For example, by Support Vector Machine an AUC of 0.804, and a sensitivity and specificity of 71.90% and 86.05 % were obtained, respectively. In other models, even before transplanting, it was also possible to predict the risk of rejection, e.g. with a Neural Network model sensitivity and specificity of 93.33% and 96.55 % were achieved, respectively. In a small cohort of patients (n=20) with rejection processes, and under immunotherapy to minimize the organ loss, it was possible to predict the efficiency of the organ rescue treatment, with e.g. Naïve Bayes or Neural Network models obtaining an AUC of 1.0 with a classification accuracy of 0.95 and 0.90 respectively. Considering the serum analysis at the time of biopsy-proven cellular (n=12) and humoral (n=42) rejection, it was not possible to develop a good prediction model, probably due to a high mix of immune rejection mechanisms. However, it was possible to identify ratios of spectral peaks based on the 2nd derivative spectra that discriminate humoral from cellular rejection (p<0.05). It was also developed an in vitro rapid test (1hr) to detect T-lymphocyte activation from the T-cells spectra that enabled the 100% discrimination, by Hierarchical Cluster Analysis of second derivative spectra, the resting T-cells from activated T-cells. Conclusions: FTIR spectroscopy of serum coupled with unsupervised and supervised processing multivariate data analysis enabled to development of good predictive models of the rejection diagnosis and prognosis, the risk of rejection before transplantation, and the efficiency of the organ rescue treatments. The basis of an in vitro method to predict T-lymphocyte activation was also developed, which could enable in the future the rapid prediction of cellular rejection processes. All these new methods, due to simplicity, speed, and economics, could increase the monitoring of these types of patients, identify critical patients with an increased risk of rejection processes, and eventually promoting the adjustment of immunotherapies for organ rescue. These could lead to disrupting modes of management of these types of patients towards a significant increase of quality of life and even of life expectancy, and in a highly economic mode.RESUMO - Enquadramento e objetivos: O transplante renal é o tratamento que proporciona a maior qualidade de vida ao insuficiente renal crónico terminal. No entanto, um dos seus principais problemas é a rejeição do aloenxerto. O presente projeto tem como objetivo promover a descoberta de biomarcadores de diagnóstico e prognóstico de processos de rejeição de aloenxertos renais e avaliar a eficiência de terapias de resgate de órgãos. Pretende-se que o diagnóstico e prognóstico seja efetuado de forma rápida, económica, mas também sensível e específico. Pretendeu-se desenvolver uma nova metodologia de descoberta de biomarcadores baseada na análise por espectroscopia de infravermelho por transformada de Fourier (FTIR) de soro associada à análise multivariada e técnicas de aprendizagem automática. Métodos: Foram incluídos 38 participantes saudáveis não transplantados, 59 pacientes transplantados renais, dos quais 12 também receberam um aloenxerto de pâncreas ou fígado. Dos pacientes transplantados, 29 apresentaram eventos de rejeição. O espectro de FTIR foi adquirido a partir de amostras de soro de não transplantados (n = 38) e transplantados sem (n = 213) e com (n = 360) rejeição. Antes da aquisição dos espectros foi otimizado o grau de diluição das amostras de soro. Foram implementados diversos métodos de análise multivariada supervisionados e não supervisionados para identificar padrões nos dados e desenvolver métodos de classificação de diagnóstico e prognóstico, de identificação do mecanismo imunológico de rejeição e a eficiência do tratamento de resgate de órgãos. Com base em amostras de sangue total de 8 voluntários saudáveis não transplantado, foi desenvolvido, um método in vitro para detetar a ativação de linfócitos T a partir de espectros de células T. Resultados: Foi possível desenvolver modelos de classificação para prever quais os pacientes que desenvolverão um processo de rejeição, por exemplo por Random Forest, foi obtida uma AUC de 0,94. Também foi possível desenvolver modelos para prever o risco de rejeição, até 120 dias antes de ser detetada em biópsia. Por exemplo, por Máquina de Vectores-Suporte obteve-se uma AUC de 0,804, e uma sensibilidade e especificidade de 71,90% e 86,05%, respetivamente. Foram desenvolvidos modelos de previsão de rejeição com base em amostras obtidas antes do transplante, por exemplo através de um modelo de Redes Neuronais foi alcançado uma sensibilidade e especificidade de 93,33% e 96,55%, respetivamente. Numa pequena coorte de pacientes (n=20) com processos de rejeição e sob imunoterapia para minimizar a perda de órgãos, foi possível prever a eficiência do tratamento de resgate do órgão, por ex. por modelos de Naïve Bayes ou de Redes Neuronais, obteve-se uma AUC de 1,0 com precisão de classificação de 0,95 e 0,90, respetivamente. Considerando a análise sérica no momento da rejeição celular (n=12) ou humoral (n=42) comprovada por biópsia, não foi possível desenvolver um bom modelo de previsão do mecanismo de rejeição com base no espectro. Foi, no entanto, possível identificar bandas espectrais com base na 2ª derivada que discriminam a rejeição humoral da celular (p<0,05). Foi desenvolvido um teste rápido in vitro para identificar ativação de linfócitos T, a partir dos espectros de células T, que implicou numa Análise Hierárquica 100% de classificação correta de linfócitos não ativados de ativados. Conclusões: A espectroscopia de FTIR do soro associada a análise multivariada, permitiu desenvolver modelos preditivos de diagnóstico e prognóstico do processo de rejeição, avaliar o risco de rejeição antes do transplante e a eficiência dos tratamentos de resgate de órgãos. Esta metodologia também permitiu desenvolver um teste in vitro, baseado na 2ª derivada do espectro de células T, para prever a ativação dos linfócitos T. Todos esses novos métodos, devido à sua simplicidade, rapidez e economia, poderão identificar pacientes críticos, com maior risco de rejeição, modificando a forma de monitorização desse tipo de paciente, e, eventualmente, promover o ajuste de imunoterapias para o resgate de órgãos, conduzir a novos modos de gestão deste tipo de pacientes que potenciaram um aumento significativo da qualidade de vida.N/