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Padronização do tempo e temperatura para determinação da atividade da enzima G6PD em amostras de papel filtro

Wed, 01 Jan 2025 00:00:00 GMT

Título: Padronização do tempo e temperatura para determinação da atividade da enzima G6PD em amostras de papel filtro Abstract: Glucose-6-phosphate dehydrogenase (G6PD) is a cytoplasmic enzyme present in all cells of the body, responsible for catalyzing the first step of the pentose phosphate pathway, which generates the cofactor NADPH. This cofactor plays a fundamental role in protecting against oxidative damage, particularly in erythrocytes, which lack alternative pathways for NADPH production. Individuals with reduced enzymatic activity are classified as G6PD-deficient. Currently, various methods are available for diagnosing G6PD deficiency, including qualitative, quantitative, semi-quantitative, and molecular tests, each with its own limitations. To ensure reliable results in enzymatic activity testing, careful control of pre-analytical variables such as time and storage conditions is essential. This study aimed to standardize the time and temperature conditions between sample collection and processing for the quantitative determination of G6PD activity in filter paper samples using a colorimetric enzymatic method. It also aimed to conduct the first population-based screening for G6PD deficiency in patients treated at Primary Health Care Units in Campo Grande, Mato Grosso do Sul, Brazil. For the standardization process, filter paper samples impregnated with whole blood were collected from 17 individuals with G6PD deficiency and 19 with normal enzymatic activity. The biological material was stored under three different conditions: room temperature, 5 °C, and -20 °C, and tested daily over a period of 15 days. For the population screening, 426 dried blood spot samples were collected and tested using a colorimetric assay to determine enzymatic activity. The results showed that room temperature caused significant instability, reducing enzymatic activity by approximately 61%. It was observed that enzyme degradation was more strongly influenced by temperature than by storage time, as accurate identification of G6PD status was no longer possible after 48 hours at room temperature. Samples stored under refrigeration remained stable for up to seven days, while those frozen at -20 °C showed even greater stability, allowing for storage of up to 14 days. Linear regression analysis indicated that 58% of the variation in G6PD enzymatic activity could be explained by storage time and temperature, suggesting that additional variables may also influence sample stability. It was concluded that storage at room temperature is a risk factor for false-positive results, and prompt analysis is recommended for such samples. Samples stored under refrigeration or freezing conditions exhibited greater stability and diagnostic reliability. In the screening performed, a prevalence of 1.1% of G6PD-deficient individuals and 15.2% with intermediate enzymatic activity was identified. Tipo: Tese

Metabolômica e redes moleculares no Rastreamento de Substâncias Bioativas de espécies vegetais: Uma Abordagem Estratégica para a Leucemia Mieloide

Wed, 01 Jan 2025 00:00:00 GMT

Título: Metabolômica e redes moleculares no Rastreamento de Substâncias Bioativas de espécies vegetais: Uma Abordagem Estratégica para a Leucemia Mieloide Abstract: Medicinal plants show a long history of use in traditional folk medicine, standing out for the diversity of properties that are beneficial to human health. They constitute important promising targets for the search for bioactive substances with potential anticancer, anti-inflammatory, antibacterial activities, among others. The Pantanal and Cerrado biomes have a wide biodiversity, sheltering numerous species of medicinal plants, whose chemical and biological properties remain little explored scientifically. In this context, advanced tools, such as metabolomics and molecular networks, have been successfully used to accelerate the discovery and reduce the time required to identify compounds of bioactive metabolites. The main objective of this study was to evaluate the cytotoxic activity of extracts obtained from eighteen plant species found in the Cerrado and Pantanal biomes of Mato Grosso do Sul against Kasumi-1, KG-1 and K562 leukemic cell lines, in addition to applying statistical analysis tools of metabolomic data together with molecular networks to determine the possible bioactive compounds. The plant samples (18 different species) were extracted by accelerated solvent extraction (ASE) using ethanol and water in the proportion 7:3 (v/v) and were analyzed by high-performance liquid chromatography coupled to a diode array detector and a high-resolution mass spectrometer (HPLC-DAD-MS) for the analysis of all extracts. The cytotoxicity of the extracts was evaluated by Alamar Blue assay and the induction of apoptosis was determined by flow cytometry with annexin V and propidium iodide, aiming to identify cells in early and late apoptosis. Metabolomic analyses, complemented by statistical analyses and molecular networks, allowed a comprehensive approach of the metabolites present in the different extracts studied, enabling the identification and analysis, allowing the annotation of steroidal and aporphinic alkaloids as priority targets. The extracts from Sesbania virgata, Aeschynomene denticulata, Erythroxylum anguifugum, Psidium guineense, Astronium fraxinifolium, Coccoloba ochreolata, Solanum glaucophyllum and Paullinia pinnata demonstrated significant inhibition of cell viability of the leukemic cell line K-562 (approximately 70% at 100 µg/mL), while Ocotea diospyrifolia showed 35% inhibition for the KG-1 cell line. Alkaloid fractions isolated from S. glaucophyllum and O. diospyrifolia demonstrated EC50 values ranging from 13.9 to 6.4 μg/mL for the K-562 and KG-1 cell lines, inducing apoptosis and significant cellular damage. The alkaloid boldine presented EC50 values of 46, 116, and 145 µM for the Kasumi-1, KG-1 and K-562 leukemia cell lines, respectively. The results highlight the importance of integrating broader analysis of chemical data for identification of bioactive compounds, emphasizing the potential benefits in the search for metabolites against leukemia cancer cells, particularly steroid and aporphine alkaloids. Therefore, metabolomic strategies and molecular networks for the effective identification of bioactive compounds, highlighting the potential of Cerrado and Pantanal plants for the development of new antileukemic agents. Tipo: Tese

Fármacos antileishmaniais: ensaios de atividade antileishmania e mecanismos de ação de complexos de usnato com íons lantanídeos e drogas de referência

Wed, 01 Jan 2025 00:00:00 GMT

Título: Fármacos antileishmaniais: ensaios de atividade antileishmania e mecanismos de ação de complexos de usnato com íons lantanídeos e drogas de referência Abstract: Leishmaniases are a group of infectious, parasitic diseases caused by parasites of the genus Leishmania sp. These diseases are endemic in several countries, including Brazil, where they pose a significant public health threat. The complexity of the disease, characterized by its diverse clinical manifestations, poses a substantial challenge in the diagnosis and treatment of leishmaniasis. Adherence to treatment regimens is hindered by adverse reactions to medications, which can lead to therapeutic failure, recurrent infections, and even fatal outcomes. Leishmania (L.) amazonensis, a prominent species in the American continent, exhibits diverse infectivity phenotypes, manifesting as anything from localised cutaneous lesions (Cutaneous Leishmaniasis, CL) to disseminated lesions, and, in more severe cases, visceralization. Previous studies using usnic acid have shown effective and promising results against species of Leishmania sp. The aim of this study was to evaluate the anti-leishmanial action of sodium usnate (soluble form of usnic acid - SAU) complexed with the lanthanide ions lanthanum - La(III), neodymium - Nd(III), gadolinium - Gd(III), terbium - Tb(III), europium - Eu(III) and samarium - Sa(III) and to clarify their possible mechanisms of leishmanicidal action. In addition, the aim was to analyse the in vitro behaviour of three strains of L. amazonensis against the reference drugs used in clinical practice, two strains isolated from patients - one from a case of CL and the other from a clinical case of visceral leishmaniasis (VL) - and one reference strain. The lanthanide complexes were highly active against promastigote forms (IC50 < 1.50 µM) and intracellular amastigotes (IC50 < 7.52 µM). The EuL3-3H2O (IC50 =2.98 µM; selectivity index, SI=6.73) and NdL3-3H2O (IC50 =2.83 µM; SI=6.97) complexes showed the highest activities, with greater selectivity in amastigote forms. In the course of investigating the mechanism of leishmanicidal action, an increase in the release of nitric oxide (NO) was detected in cells infected with L. amazonensis and treated with EuL3·3H2O (6.25 µg/mL). This finding suggests that this may be one of the mechanisms involved. Furthermore, alterations in mitochondrial membrane potential were evident in parasites exposed to SmL3-4H2O and GdL3-2H2O, suggesting that lanthanides complexed with sodium usnate can enhance their anti-leishmania activity, with the parasite's mitochondria serving as the primary target. With respect to the efficacy of the reference drugs on the various strains of L. amazonensis, it is noteworthy that Amphotericin B exhibited high activity against both evolutionary forms of the parasites. Pentamidine demonstrated notable activity against the MHOM/BR/2022/LV045_22 strain (IC50 = 0.0429 µM), which was isolated from a patient with visceral leishmaniasis (VL), underscoring the potential of pentamidine in the development of technologies aimed at mitigating adverse effects to enhance treatment efficacy. Tipo: Tese

Síntese de flavonoides e avaliação da atividade inibitória sobre a COVID-19

Wed, 01 Jan 2025 00:00:00 GMT

Título: Síntese de flavonoides e avaliação da atividade inibitória sobre a COVID-19 Abstract: In late 2019, a viral outbreak originating in Wuhan, China, led to the COVID-19 pandemic, presenting unprecedented challenges to global public health. While agencies such as the U.S. Food and Drug Administration (FDA) and Brazil's National Health Surveillance Agency (ANVISA) have authorized certain treatments for COVID-19, these therapies often come with limitations, including drug interactions, the necessity for intravenous administration, and limited efficacy. Consequently, there is a pressing need for the development of more effective and accessible antiviral agents. In response to this need, collaborations between the pharmaceutical industry and academic institutions have increasingly employed Computer-Aided Drug Design (CADD). This approach facilitates the creation of bioactive compounds by analyzing the structures of molecular targets. A notable example is nafamostat mesylate, a serine protease inhibitor traditionally used for other indications, which has demonstrated the ability to inhibit SARS-CoV-2 replication by targeting the TMPRSS2 enzyme. Additionally, secondary metabolites such as flavonoids and capsaicinoids have shown potential antiviral properties. Flavonoids, widely found in various plants, exhibit antioxidant, anti-inflammatory, and antiviral activities. Capsaicinoids, have also demonstrated inhibitory effects against SARS-CoV-2. Given the significant health and economic impacts of the COVID-19 pandemic, this study aimed to synthesize compounds with potential antiviral activity using CADD methodologies. The proposed synthetic routes were designed to be straightforward, sustainable, and scalable. The study is organized into three chapters: the first discusses the pathology of SARS-CoV-2 and the global health emergency it precipitated; the second details the synthesis of capsaicin analogs derived from nafamostat mesylate; and the third presents findings related to flavonoid analogs and prospects for COVID-19. Tipo: Dissertação