Cândida Tomaz

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Chronic heart failure hampers regulatory T cell homeostasis which is not restored by cardiac resynchronization therapy

Background: T cells have been implicated in the development and progression of in ammatory proces... more Background: T cells have been implicated in the development and progression of in ammatory process in chronic heart failure (CHF). Cardiac resynchronization therapy (CRT) has bene cial effects on symptoms and cardiac remodeling in CHF. However, its impact on the in ammatory immune response remains controversial. We aimed to study the impact of CRT on T cells in heart failure patients (HFP). Methods: Thirty-nine HFP were evaluated before CRT (T0) and 6 months later (T6). Quanti cation of T cells, their subsets, and their functional characterization, after in vitro stimulation, were evaluated by ow cytometry. Results: T regulatory (Treg) cells were decreased in CHF patients (healthy group (HG): 1.08±0.50 versus HFP-T0: 0.69±0.40, P=0.022) and remaining diminished after CRT (HFP-T6: 0.61±0.29, P=0.003). Responders (R) to CRT presented a higher frequency of T cytotoxic (Tc) cells producing IL-2 at T0 compared with non-responders (NR)

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G-Quadruplex Aptamer-Ligand Characterization

Molecules

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGG... more In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the additi...

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Comparison of Vascular Endothelial Growth Factors (VEGFs) A and B with diabetic retinopathy severity and diabetic macular edema. Correlation between growth factors and anatomical parameters

Investigative Ophthalmology & Visual Science, 2021

Processes for reducing egg allergenicity: Advances and different approaches

Food Chemistry, 2022

Egg is a versatile ingredient and ubiquitous food. Nevertheless, egg proteins are a common cause ... more Egg is a versatile ingredient and ubiquitous food. Nevertheless, egg proteins are a common cause of allergy mainly in childhood. Until now, egg eviction has been the best way to prevent this disorder, however, processed food can contribute to mitigate allergies and to guarantee life quality of allergic individuals. This review focuses on discussing and highlighting recent advances in processes to reduce egg allergenicity as well as new approaches to egg allergy management. In recent times, different methods have been developed to reduce egg allergies, by hiding the epitopes or changing the native or conformational structure of the proteins. Despite processing food has not yet been a solution to completely remove the allergenic potential of egg proteins, innovative strategies, such as addition of phenolic compounds, have been developed with promising results.

Ovalbumin Structural Changes by Phenolic Compounds Interactions

Ovalbumin (OVA) is the most prevalent protein in egg white and, represents the major allergen fro... more Ovalbumin (OVA) is the most prevalent protein in egg white and, represents the major allergen from avian egg white that causes IgE-mediated food allergic reactions particularly in children. It has been shown that phenolic compounds interact with proteins by a single or multipoint mechanism, promoting structural and functional changes. Moreover, the interaction of some allergens with polyphenols, led to permanent modification of the tertiary structure of the allergen, which can result in a reduction in its IgE-binding capacity. This work aimed to analyse the effect of phenolic compounds (Gallic, Caffeic, Ferulic, Chlorogenic and Tannic Acids, Resveratrol and Quercetin) on the native structure of OVA, using Circular Dichroism (CD), fluorescence and Fourier transform infrared spectroscopy (FTIR). The phenolic compounds were incubated with OVA at different times, concentrations and temperatures. Changes in OVA secondary and tertiary structures were increasingly induced with increasing temperatures by the phenolic compound. Also, for a constant temperature, the changes found in OVA structure increased with the phenolic compounds concentration. The results show that the interactions between phenolic compounds and OVA result in complexes (phenolics-OVA) where OVA native structure is changed. This is likely to affect epitopes and hence OVA allergenicity.

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Quantification and comparison of VEGF-B in the vitreous of patients with diabetic ocular disease and a control group of patients with non-diabetic ocular disease

Investigative Ophthalmology & Visual Science, 2014

Comparison of serum and vitreous PIGF in diabetic retinopathy patients and non-diabetic patients

Investigative Ophthalmology & Visual Science, 2015

Interactions of Phenolic Compounds with Ovalbumin

It is well known that phenolic compounds can bind to proteins promoting structural and functional... more It is well known that phenolic compounds can bind to proteins promoting structural and functional changes. In this work, the interactions between OVA and the phenolic compounds were studied through spectroscopic techniques (fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR)) and docking. OVA solutions were incubated at different temperatures and times, with different phenolic compounds prepared with the same buffer solution (Gallic, Caffeic, Ferulic, Chlorogenic and Tannic Acids, Resveratrol and Quercetin). Results indicate that OVA's structure was affected by the binding of phenolic compounds. CD and FTIR experiments showed changes in the secondary structure of OVA, originated by the conversion of α-helix into β-sheets [1]. Fluorescence spectra demonstrated that phenolics are quenchers of fluorescent aminoacids (Tyrosine, Phenylalanine and Tryptophan) meaning that interactions occur directly or near these aminoacids. Fluorescence results also ...

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Phenolic compounds: a novel approach to reduce egg allergenicity?

Hen's egg allergy has been climbing in all countries due to eggs ubiquity in foodstuffs. This... more Hen's egg allergy has been climbing in all countries due to eggs ubiquity in foodstuffs. This allergy is an IgE mediated reaction that affects mainly infants and young children. So far, the processes used to decrease the allergenicity of egg proteins, such as cooking, thermal processing, storage and enzymatic digestion have not been totally effective. Previous studies demonstrated that proteins form complexes with phenolic compounds, so the aim of this work was to analyze the effect of these compounds in ovalbumin (OVA) conformation and its possible application to reduce eggs allergenicity. OVA was incubated at different temperatures with phenolic compounds (caffeic, chlorogenic, ferulic, gallic and tannic acids; resveratrol and quercetin) and was analyzed by circular dichroism (CD), Attenuated Total Reflection–Fourier Transform Infra-Red (ATRFTIR) spectroscopy and fluorescence. Changes in the secondary structure of OVA were evidenced by CD and ATR-FTIR. Also, protein fluorescen...

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Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection

Sensors, 2021

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays ... more The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with ...

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Evaluation of the growth factors VEGF-a and VEGF-B in the vitreous and serum of patients with macular and retinal vascular diseases

Growth Factors, 2018

VEGF-A and VEGF-B are proangiogenic and key regulating factors for blood vessel growth. This stud... more VEGF-A and VEGF-B are proangiogenic and key regulating factors for blood vessel growth. This study aims to compare VEGF-A and VEGF-B levels in the serum and vitreous of patients with neovascular pathology versus non-neovascular pathology. Our findings showed vitreous VEGF-A and VEGF-B levels increased in patients with neovascular disease, with higher levels of VEGF-A compared to VEGF-B (p .05). In the diabetic retinopathy (DR) group, higher vitreous VEGF-A or VEGF-B were found in proliferative diabetic retinopathy (PDR) than in non-PDR. The strong correlation between VEGF-A and VEGF-B demonstrates a simultaneous pathological increase of cytokines (p < .001), suggesting besides VEGF-A, VEGF-B is another contributor to ocular pathologies involving angiogenesis. There was no correlation between vitreous and serum VEGF-A or VEGF-B; however, a correlation between vitreous (VEGF-A or VEGF-B) and macular volume (p < .05) in DR patients was found. Targeting VEGF-A and VEGF-B in macular and retinal vascular diseases, involving neovascularization, may improve treatment outcomes.

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ESBL and AmpC β-Lactamases in Clinical Strains of Escherichia coli from Serra da Estrela, Portugal

Medicina, 2019

Background and Objectives: Given the considerable spatial, temporal, and ecological factors, hete... more Background and Objectives: Given the considerable spatial, temporal, and ecological factors, heterogeneity, which affects emergency response, persistence, and dissemination of genetic determinants that confer microorganisms their resistance to antibiotics, several authors claim that antibiotics’ resistance must be perceived as an ecological problem. The aim of this study was to determine the prevalence of broad-spectrum bla genes, not only Extended-spectrum β-lactamases (ESBL) but also AmpC-types, in clinical strains of Escherichia coli isolated from Portugal (in the highest region of the country, Serra da Estrela) to disclose susceptibility profiles among different genotypes, and to compare the distribution of bla genes expressing broad-spectrum enzymes. Materials and Methods: Clinical strains of Escherichia coli presenting resistance to third generation (3G) cephalosporins and susceptibility to inhibition by clavulanic acid were studied by means of phenotypic and molecular profili...

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Vascular endothelial growth factors and placenta growth factor in retinal vasculopathies: Current research and future perspectives

Cytokine & Growth Factor Reviews, 2018

B and PlGF in the pathophysiology and pathogenesis of various ophthalmic disorders. ► A discussio... more B and PlGF in the pathophysiology and pathogenesis of various ophthalmic disorders. ► A discussion of investigational studies of human vitreous and serum VEGF-A, VEGF-B and PIGF and importance in diagnostic and therapy. ► An analysis of current targeted therapies for retinal diseases. ► A reflection on new promising molecules in ophthalmology.

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Influenza DNA vaccine purification using pHEMA cryogel support

Separation and Purification Technology, 2018

Influenza virus is a huge financial and social burden for health care systems over the world. Cur... more Influenza virus is a huge financial and social burden for health care systems over the world. Currently, traditional approaches are not effective in the fight of the epidemy and new alternatives like DNA vaccines have been developed. However, the downstream process of DNA vaccines is a constant challenge in the biotechnology industry. Cryogels has several advantages over traditional supports and have been tested as stationary phase in chromatographic separations. In this work, a method based on poly(2-hydroxyethyl methacrylate) cryogel was used to purify the plasmid NTC7482-41H-VA2 HA, which express the Influenza hemagglutinin gene. For this purpose, the cryogel was synthesized by cryo-polymerization of 2-hydroxyethyl methacrylate and characterized by scanning electron microscopy. The purification of supercoiled isoform of the plasmid NTC7482-41H-VA2 HA from a clarified lysate sample was achieved in a two-step experiment using NaCl and the dynamic binding capacity of pHEMA cryogel was determined. The assessment of DNA vaccine allowed to conclude that the level of contaminants such as proteins, genomic DNA, RNA and endotoxins are in accordance with FDA agency.

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iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

International journal of molecular sciences, Jan 11, 2018

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a ph... more Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator...

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VEGF-B Levels in the Vitreous of Diabetic and Non-Diabetic Patients with Ocular Diseases and Its Correlation with Structural Parameters

Medical Sciences, 2017

Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family.... more Vascular endothelial growth factor B (VEGF-B) is one of the enigmatic members of the VEGF family. The knowledge gap about VEGF-B expression and how its levels are altered in diabetic eyes were the focus of this investigation that was addressed by comparing and correlating vitreous VEGF-B between diabetic and non-diabetic patients. VEGF-B levels were measured by enzyme-linked immunosorbent assay in vitreous samples (n = 33) from diabetic (n = 25) and non-diabetic (n = 8) patients. Results were compared between groups. Optical coherence tomography from diabetic patients was evaluated for central retinal thickness (CRT) and macular volume (MV). Mean vitreous VEGF-B concentration was higher in diabetic (18.82 ± 1.44 pg/mL) vs. non-diabetic patients (17.90 ± 0.32 pg/mL) (p = 0.006), and in proliferative diabetic retinopathy (PDR) (19.03 ± 1.52 pg/mL) vs. non-PDR (NPDR) patients (18.18 ±0.96 pg/mL) (p = 0.025). In diabetic retinopathy (DR) patients, correlation between VEGF-B and CRT (µm) was positive and moderate: r s = 0.441 (p ≤ 0.05) and the correlation between VEGF-B and MV (mm 3) was positive and robust: r s = 0.716 (p ≤ 0.01). VEGF-B levels are overexpressed in vitreous of diabetic patients, and the levels are higher in developed stages of DR. Correlation results show that CRT and MV increase with increased levels of VEGF-B. Targeting VEGF-B inhibition may have therapeutic beneficial implications.

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$Research paper thumbnail of Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation$

Proteome analysis of vitreous humor in retinal detachment using two different flow-charts for protein fractionation

Journal of Chromatography B, 2017

Highlights  Two fractionation strategies were proposed for the vitreous proteome analysis in ret... more Highlights  Two fractionation strategies were proposed for the vitreous proteome analysis in retinal detachment.  A total of 127 proteins were identified in vitreous of patients with retinal detachment.  From the proteins identified in vitreous, 68 had not yet been found in previous studies.  Bidimensional fractionation is more suitable to increase the coverage of vitreous proteome.

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Alternaria alternata allergens: Markers of exposure, phylogeny and risk of fungi-induced respiratory allergy

Environment international, Jan 27, 2016

Alternaria alternata spores are considered a well-known biological contaminant and a very common ... more Alternaria alternata spores are considered a well-known biological contaminant and a very common potent aeroallergen source that is found in environmental samples. The most intense exposure to A. alternata allergens is likely to occur outdoors; however, Alternaria and other allergenic fungi can colonize in indoor environments and thereby increase the fungal aeroallergen exposure levels. A consequence of human exposure to fungal aeroallergens, sensitization to A. alternata, has been unequivocally associated with increased asthma severity. Among allergenic proteins described in this fungal specie, the major allergen, Alt a 1, has been reported as the main elicitor of airborne allergies in patients affected by a mold allergy and considered a marker of primary sensitization to A. alternata. Moreover, A. alternata sensitization seems to be a triggering factor in the development of poly-sensitization, most likely because of the capability of A. alternata to produce, in addition to Alt a 1...

Purification of influenza deoxyribonucleic acid-based vaccine using agmatine monolith

Journal of Chromatography B, 2016

Lately, researchers have made several efforts to improve vaccine production to fight highly conta... more Lately, researchers have made several efforts to improve vaccine production to fight highly contagious respiratory diseases like influenza. One of the most promising options for reducing the impact of this virus is DNA vaccination. However, a large quantity of highly pure plasmid DNA (pDNA) is necessary to attain this goal. The present work describes the production and purification of the plasmid NTC7482-41H-VA2HA expressing influenza virus hemagglutinin using an agmatine monolith. This ligand was chosen to purify supercoiled (sc) pDNA from complex lysates because of its versatile multimodal character. Its natural intervention in several biological systems together with its similarity with the highly studied arginine ligand allowed the development of a simpler and more specific purification process. Agmatine works under two strategies: descending ammonium sulfate gradient and ascending sodium chloride gradient. Furthermore, pH manipulation revealed an important role in pDNA isoforms selectivity. Dynamic binding capacity (DBC) experiments were performed varying different parameters and showed an increase with pDNA concentration, while high flow rate and high pH had the opposite effect. Sc pDNA was purified with high yield and was efficient with respect to cell transfection and cell viability. This monolith showed to be appropriate to purify the plasmid NTC7482-41H-VA2HA, providing a valuable tool for pDNA influenza vaccines preparation.

Influenza plasmid DNA vaccines: progress and prospects

Current Gene Therapy, 2015

Current influenza vaccines have long been used to fight flu infectious; however, recent advances ... more Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

Cândida Tomaz

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