Novas abordagens para o tratamento da fenilcetonúria: avaliação em modelos celulares de formulações da fenilcetonúria hidroxilase humana

Tese de mestrado, Bioquímica (Bioquímica Médica) Universidade de Lisboa, Faculdade de Ciências, 2016A Fenilcetonúria (PKU; OMIM 261000) é o erro hereditário mais comum do metabolismo dos aminoácidos afetando, na população Caucasiana, cerca de 1 em cada 10.000 recém-nascidos. Esta doença genética tem como base mutações no gene que codifica para a fenilalanina hidroxilase humana (hPAH; EC 1.14.16.1), uma enzima que catalisa a hidroxilação da fenilalanina (L-Phe) em tirosina (LTyr) na presença do cofator tetra-hidrobiopterina (BH4) e do oxigénio molecular (O2). De modo a evitar danos cerebrais irreversíveis deve ser instaurada uma dieta pobre no aminoácido essencial L-Phe o mais cedo possível após o nascimento. Até muito recentemente a terapia dietética era a única abordagem disponível para o tratamento dos doentes PKU. Uma vez que a PKU pode ser considerada uma doença conformacional por loss of function nos últimos anos surgiu uma alternativa terapêutica que envolve administração do cofator enzimático BH4 como chaperone farmacológico. No entanto, estas duas abordagens terapêuticas estão longe de abranger todo o espectro fenotípico da doença, em especial o fenótipo de PKU clássica, mais grave. Os grupos Metabolism and Genetics (Met&Gen) e Nanostructured Systems for Overcoming Biological Barriers (Nano2B) do Research Institute for Medicines (iMed.ULisboa), têm estado envolvidos no desenvolvimento de uma nova abordagem terapêutica designada por terapia enzimática de reposição (ERT). Neste âmbito foi já possível obter uma forma estável da hPAH e desenvolver um sistema de nanopartículas (NP) de quitosano (CS) adequado para a encapsulação da forma selvagem da hPAH (hPAHwt). Assim, o presente trabalho teve como objetivo verificar a eficácia das formulações obtidas por ensaios de uptake em células apropriadas nomeadamente, células HEK-293T (human embryonic kidney 293T cells). Uma vez que estas células não expressam hPAH a aquisição de capacidade hidroxilante da L-Phe no lisado celular poderá confirmar o sucesso das formulações desenvolvidas. Assim, a proteína hPAHwt foi produzida num sistema de expressão procariota e a sua forma tetramérica biologicamente ativa foi isolada utilizando técnicas de cromatografia de afinidade e de exclusão molecular. A proteína recombinante produzida foi caraterizada a nível da sua atividade enzimática e estabilidade térmica (Tm) por fluorimetria diferencial de varrimento (DSF). De entre as formulações previamente otimizadas no laboratório, foi selecionada a formulação de NP-CS e cinco outras formulações onde foram utilizados diferentes agentes modificadores. Estes, por questões de confidencialidade, foram designados por 1, 2, 3, 4 e 5 por (NP-CSAgm1, NP-CSAgm2, NP-CSAgm3, NP-CSAgm4 e NP-CSAgm5). Para preparação das NPs com os agentes modificadores 1, 2 e 3 foram ainda ensaiadas cargas baixas (Agmcb) e altas (Agmca). Após caraterização das NPs e do perfil de libertação das formulações em estudo foram então efetuados os ensaios em células. Após nanoencapsulção da hPAHwt nos diferentes sistemas, a avaliação da atividade catalítica e do Tm permitiu confirmar que esta mantinha a sua funcionalidade (74,3 ± 5,7% de atividade relativa) e estabilidade. A caracterização das NPs indicaram que estas apresentavam propriedades adequadas à sua utilização no que respeita ao tamanho de partícula (221 ± 24 nm), potencial zeta (+26,1 ± 2,4 mV) e eficiência de encapsulação (EE; 78,6 ± 2,8%). Os ensaios de libertação efetuados demonstraram que todas as formulações, exceto a NP-CSAgm2ca, mantinham constantes o valor de proteína no sobrenadante dos ensaios. Assim, de entre as nove formulações foram selecionadas quatro (NP-CS, NPCSAgm3ca, NP-CSAgm4 e NP-CSAgm5) para os ensaios com células HEK-293T. Uma vez que não foi possível detetar hPAHwt na fração solúvel do lisado celular estendemos os nossos ensaios às células HepG2 (human hepatocellular carcinoma cell line, ATCC HB-8065). De entre as formulações testadas duas apresentaram capacidade de serem internalizadas pelas células HepG2 nomeadamente a NP-CS e a NP-CSAgm5 uma vez que o teor de hPAHwt detetado no sobrenadante do lisado, após 6 h de incubação a 37 °C, foi de 26,2% e 8,2%, respetivamente. Os resultados obtidos neste trabalho são extremamente promissores e abrem a porta a novos estudos que poderão ter como objetivo a modificação da superfície das NPs para melhorar o seu uptake celular.Phenylketonuria (PKU; OMIM 261000) is the most frequent inborn error of amino acid metabolism with an estimated incidence of 1:10,000 newborns, in the Caucasian population. This genetic disease is the result of mutations in the gene encoding the human phenylalanine hydroxylase (hPAH; EC 1.14.16.1). This enzyme is responsible for the hydroxylation of L-phenylalanine (L-Phe) into L-tyrosine (L-Tyr) in the presence of the tetra-hydrobiopterin cofactor (BH4) and the molecular dioxygen (O2). In order to avoid irreversible neurological damages a diet restricted in L-Phe should be implemented as soon as possible after birth. Till very recently this diet was the only available approach to PKU treatment. As PKU is now classified as a conformational disorder with loss of function, in the last years an alternative therapeutic approach was developed involving BH4 administration as a pharmacological chaperone. However, these therapeutic approaches are far from including the full phenotypic spectra of the disease and in particular the most severe classical form. The groups of Metabolism and Genetics (Met&Gen) and Nanostructured Systems for Overcoming Biological Barriers (Nano2B), from the Research Institute for Medicines (iMed.ULisboa), have been involved in the development of an enzyme reposition therapy (ERT) as a new approach to PKU treatment. In this scope the groups already obtained a stable form of hPAH and developed a chitosan (CS) nanoparticulate (NP) system allowing an efficient encapsulation of the wild-type hPAH (hPAHwt). As such, this work aimed at verify the efficacy of the designed formulations by performing uptake assays in appropriate cells namely HEK-293T cells (human embryonic kidney 293T cells). Since, hPAH is not expressed in these cells acquisition of a L-Phe hydroxylating capacity by the cellular lysate should confirm the success of the developed formulations. To this end, the hPAHwt was produced in a prokaryotic expression system and the biological active tetrameric form was purified using two chromatographic techniques namely affinity chromatography and size exclusion chromatography. The obtained recombinant protein was characterized concerning its catalytic activity and thermal stability (Tm) using differential scanning fluorimetry (DSF). Among the formulations previously optimized in our laboratory, we selected for further cellular studies the NP-CS and five additional formulations prepared using modifying agents. These agents were named 1, 2, 3, 4 and 5 (NP-CSAgm1, NP-CSAgm2, NP-CSAgm3, NP-CSAgm4 and NP-CSAgm5) due to confidentiality issues. When using the modifying agents 1, 2 and 3 a low (Agmcb) and a high (Agmca) loading were also tested. After characterization of NPs and their release profile cellular studies were then performed. Upon hPAHwt nanoencapsulation using the nine different systems, evaluation of the specific enzymatic activity and Tm allowed to confirm that the protein maintained functionality (74.3 ± 5.7% of relative enzyme activity) and stability. The NPs physicochemical characterization showed that the formulations presented adequate properties to be further used in the cellular assays in respect to NP size (221 ± 24 nm), zeta potential (+26.1 ± 2.4 mV) and encapsulation efficiency (EE; 78.6 ± 2.8%). The release assays indicated that all but one (NP-CSAgm2ca) of the tested formulations allowed to maintain a constant value of the protein in the supernatant of the assays. Therefore, among these nine formulations four (NP-CS, NP-CSAgm3ca, NP-CSAgm4 e NP-CSAgm5) were selected for further HEK-293T cellular assays. Since, no hPAHwt protein was detected in the soluble fraction of the cellular lysate additional assays were performed using HepG2 cells (human hepatocellular carcinoma cell line, ATCC HB-8065). Among the four formulations studied, two showed to present the capacity to be internalized by the HepG2 cells namely NP-CS and NP-CSAgm5 as the content of the hPAHwt protein detected in the supernatant of the cellular lysate, after 6 h incubation at 37 °C, was 26.2% and 8.2%, respectively. The results obtained in this work are very promising and pave the way to additional studies aiming to modify the NP surface in order to improve their cellular uptake

Conservação de alimentos por exposição à radiação ionizante

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2020, Universidade de Lisboa, Faculdade de Farmácia.Encontram-se atualmente descritas na literatura várias técnicas de conservação de alimentos, tendo todas elas o objetivo de aumentar o tempo de vida útil dos mesmos, evitando perdas nutricionais consideráveis. Uma das abordagens da Indústria alimentar tem consistido na utilização da energia ionizante para efeitos da conservação dos produtos alimentícios. A irradiação é um método físico que se baseia na exposição do alimento a uma determinada quantidade de radiação ionizante e por um determinado intervalo de tempo, tendo em conta as características do alimento a ser processado e a finalidade pretendida com a aplicação da técnica. O objetivo do presente trabalho foi realizar um levantamento bibliográfico com o intuito de fazer uma avaliação do panorama geral dos efeitos do uso da radiação ionizante nos microrganismos e componentes nutricionais dos alimentos, suas vantagens, princípios e limitações. A irradiação é utilizada de forma a minimizar a flora microbiana e a diminuir a velocidade das reações químicas intrínsecas dos alimentos. No âmbito da conservação dos alimentos, os tipos de radiação envolvidos são os raios gama (os mais utilizados), os raios X e os eletrões de elevada energia. A preocupação dos consumidores em relação aos alimentos processados por esta técnica é considerada uma limitação na difusão da mesma. Não obstante, os vários estudos reportados na literatura têm vindo a comprovar que o método de irradiação dos alimentos continua a ter um potencial promissor para o futuro, tornando-se fundamental a correta divulgação das suas vantagens e segurança dos métodos de irradiação.Nowadays we can find several methods of food preservation and all of them share a common goal which is to increase food durability, avoiding unnecessary loss of nutrient components. One of the approaches of the Food Industry has been the use of ionizing energy for the purpose of preserving food products. Irradiation is a physical principle based on exposing a food product to a certain amount of ionizing radiation during a defined amount of time, taking into account the characteristics of the product itself and our intended purpose. The main objective of this thesis was to gather all the existing bibliographic information on the matter, to evaluate the effects of the use of ionizing radiation in the microorganisms and nutrient components of food products, and also its advantages, principles and limitations. Ionizing radiation is applied to minimize microbial flora and reduce the speed of food intrinsic chemical reactions. In the context of food preservation, the types of radiation involved are gamma rays (the most used), X-rays and high-energy electrons. The consumer’s concern about the use of this technique in food preservation is largely assumed as a limitation to propel it further in the market. However, recent studies show that preserving food products by means of radiation has a promising future and therefore it is imperative to correctly disclose it, in regard to its advantages and safety, to minimize consumer’s misconceptions

Analysis of the seroprevalence of and factors associated with Chagas disease in an endemic area in Northeastern Brazil

ABSTRACT INTRODUCTION: Chagas disease (CD) is currently considered a neglected disease; hence, identifying the factors associated with its high prevalence is essential. This study aimed to identify the seroprevalence of and the possible factors associated with CD in inhabitants of the City of Limoeiro do Norte, Northeastern Brazil. METHODS: Between April and November 2013, blood collection was conducted and a semi-structured questionnaire was administered. Blood samples that showed positive or possible serology for anti-Trypanosoma cruzi antibodies based on indirect immunofluorescence, hemagglutination indirect, and an enzyme-linked immunosorbent assay were analyzed. Associations between CD positivity and the study variables were analyzed using prevalence ratios (PR) with 95% confidence intervals (CI). RESULTS: A total of 812 individuals were analyzed, of which T. cruzi seropositivity was determined in 4.2% (34 individuals). Sociodemographic variables showing a significant association with T. cruzi positivity included age >50 years (PR = 27.6; 95% CI = 6.66-114.4), elementary level education (PR = 5.15; 95% CI = 1.83-14.47), and retirement (PR = 7.25; 95% CI = 3.72-14.14). Positivity for T. cruzi was 6.17 times higher in those who had a history of living in rammed earth houses compared with those who did not (95% CI = 2.19-17.37). There was no evidence of vertical transmission in the individuals studied. Among the individuals infected with T. cruzi, the majority reported having a comorbidity (p < 0.01). CONCLUSIONS: This study demonstrated the seroprevalence of CD and identified factors associated with a high prevalence of CD

Insect vectors of Chagas disease (Trypanosoma cruzi) in Northeastern Brazil

Abstract INTRODUCTION: Chagas disease remains a public health problem in the rural and urban areas of 19 countries in the Americas. METHODS: The aim of the present study was to investigate the Trypanosoma cruzi infection rate of triatomines collected from both intra- and peridomiciliary areas in eleven municipalities of Southeastern Ceará, Brazil, from 2009 to 2015. RESULTS: A total of 32,364 triatomine specimens, including nymphs and adults, were collected, and 31,736 (98.06%) of these were examined. More nymphs were collected than adults, and the greatest number of triatomines (n = 8,548) was collected in 2010, for which the infection rate was 1.3%, with the highest rate of infections observed for specimens from Quixere. The species collected during the study were identified as Triatoma pseudomaculata, Triatoma brasiliensis, Panstrongylus megistus, Panstrongylus lutzi, and Rhodnius nasutus, with T. pseudomaculata being the most abundant (n = 19,962). CONCLUSIONS: These results verify the presence of triatomines in both intra- and peridomiciliary areas, thereby ensuring persistence of the pathogen and consequently, the disease, as the presence of infected vectors in households is an important risk factor. According to these findings, the Chagas Disease Control Program should intensify its efforts in order to prevent the spread of the disease

Núcleos de Ensino da Unesp: artigos 2007

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Núcleos de Ensino da Unesp: artigos 2008

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq