CARLOS ROBERTO JORGE SOARES

CARLOS ROBERTO JORGE SOARES

(Fonte: Lattes)

Resumo

Graduado em Farmácia e Bioquímica pela Universidade de São Paulo (1989), realizou Mestrado (1995) e Doutorado (2000) em Tecnologia Nuclear - Aplicações pela Universidade de São Paulo. Atualmente é pesquisador do Instituto de Pesquisas Energéticas (IPEN-CNEN/SP) e professor de pós-graduação vinculado à Universidade de São Paulo. Com experiência em biotecnologia na expressão de proteínas recombinantes por bactéria e por células de mamífero. Atua principalmente no seguinte tema: síntese, purificação, caracterização e aplicações de hormônios recombinantes. Atualmente é Gerente do Centro de Biotecnologia do IPEN. (Texto extraído do Currículo Lattes em 08 out. 2021)

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Estabelecimento de um consórcio entre CEMIB, IPEN e Instituto Butantan, para a capacitação, implantação e desenvolvimento da técnica de criopreservação e reprodução assistida de animais de laboratório
2023 - MATTARAIA, VANIA G. de; SALGADO, ANDREIA R.; DEMOLIN, DANIELA M.R.; MONTEIRO, KARIN M.; SOARES, CARLOS R.J.; SPENCER, PATRICK J.; MARQUEZI, CYNTHIA Z. de A.; PASSOS, LUIZ A.C.
Isolation and characterization of the Arapaima gigas growth hormone (ag-GH) cDNA and three-dimensional modeling of this hormone in comparison with the human hormone (hGH)
2023 - LIMA, ELIANA R.; FREIRE, RENAN P.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; YOSIDAKI, VANESSA L.; PERONI, CIBELE N.; SEVILHANO, THAIS; ZORZETO, MOISES; TORATI, LUCAS S.; SOARES, CARLOS R.J.; LIMA, IGOR D. de M.; KRONENBERGER, THALES; MALTAROLLO, VINICIUS G.; BARTOLINI, PAOLO
In a previous work, the common gonadotrophic hormone α-subunit (ag-GTHα), the ag-FSH β- and ag-LH β-subunit cDNAs, were isolated and characterized by our research group from A. gigas pituitaries, while a preliminary synthesis of ag-FSH was also carried out in human embryonic kidney 293 (HEK293) cells. In the present work, the cDNA sequence encoding the ag-growth hormone (ag-GH) has also been isolated from the same giant Arapaimidae Amazonian fish. The ag-GH consists of 208 amino acids with a putative 23 amino acid signal peptide and a 185 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the Elopiformes (82.0%), followed by Anguilliformes (79.7%) and Acipenseriformes (74.5%). The identity with the corresponding human GH (hGH) amino acid sequence is remarkable (44.8%), and the two disulfide bonds present in both sequences were perfectly conserved. Three-dimensional (3D) models of ag-GH, in comparison with hGH, were generated using the threading modeling method followed by molecular dynamics. Our simulations suggest that the two proteins have similar structural properties without major conformational changes under the simulated conditions, even though they are separated from each other by a >100 Myr evolutionary period (1 Myr = 1 million years). The sequence found will be used for the biotechnological synthesis of ag-GH while the ag-GH cDNA obtained will be utilized for preliminary Gene Therapy studies.
Molecular cloning and AlphaFold modeling of thyrotropin (ag-TSH) from the Amazonian fish Pirarucu (Arapaima gigas)
2023 - FREIRE, RENAN P.; HERNANDEZ-GONZALEZ, JORGE E.; LIMA, ELIANA R.; SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E. de; TORATI, LUCAS S.; BARTOLINI, PAOLO; SOARES, CARLOS R.J.
Arapaima gigas, known as Pirarucu in Brazil, is one of the largest freshwater fish in the world. Some individuals could reach 3 m in length and weight up to 200 kg. Due to extinction risks and its economic value, the species has been a focus for preservation and reproduction studies. Thyrotropin (TSH) is a glycoprotein hormone formed by 2 subunits α and β whose main activity is related to the synthesis of thyroid hormones (THs)—T3 and T4. In this work, we present a combination of bioinformatics tools to identify Arapaima gigas βTSH (ag-βTSH), modeling its molecular structure and express the recombinant heterodimer form in mammalian cells. Using the combination of computational biology, based on genome-related information, in silico molecular cloning and modeling led to confirm results of the ag-βTSH sequence by reverse transcriptase-polymerase chain reaction (RT-PCR) and transient expression in human embryonic kidney (HEK293F) cells. Molecular cloning of ag-βTSH retrieved 146 amino acids with a signal peptide of 21 amino acid residues and 6 disulfide bonds. The sequence has a similarity to 39 fish species, ranging between 43.1% and 81.6%, whose domains are extremely conserved, such as cystine knot motif and N-glycosylation site. The Arapaima gigas thyrotropin (ag-TSH) model, solved by AlphaFold, was used in molecular dynamics simulations with Scleropages formosus receptor, providing similar values of free energy ΔGbind and ΔGPMF in comparison with Homo sapiens model. The recombinant expression in HEK293F cells reached a yield of 25 mg/L, characterized via chromatographic and physical-chemical techniques. This work shows that other Arapaima gigas proteins could be studied in a similar way, using the combination of these techniques, recovering more information from its genome and improving the reproduction and preservation of this prehistoric fish.
In vitro cytotoxic data on Se-methylselenocysteine conjugated to dendritic poly(glycerol) against human squamous carcinoma cells
2022 - CORREA, NICOLI D.G.; SILVA, FELIPE D.; VIEIRA, DANIEL P.; SOARES, CARLOS R.J.; QUEIROZ, ALVARO A.A. de
Polymeric nanoparticles acting as sources of selenium (Se) are currently an interesting topic in cancer chemotherapy. In this study, polyglycerol dendrimer (DPGLy) was functionalized with seleno-methyl-selenocysteine (SeMeCys) by means of Steglich esterification with 4-dimethylaminopyridine/(l-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (EDC/DMAP) and cerium chloride as cocatalyst in acetonitrile at quantitative yields of 98 ± 1%. The SeMeCys coupling DPGLy efficiency vs. time were determined by Fourier Transform infrared spectroscopy (FTIR) and ultraviolet–visible (UV–Vis) spectroscopy. The cytotoxic effects of SeMeCys–DPGLy on the Chinese Hamster ovary cell line (CHO-K1) and head and neck squamous cell carcinoma (HNSCC) cells line were assessed by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. No signs of general toxicity of SeMeCys–DPGLy against CHO-K1 cells were detectable at which cell viability was greater than 98%. MTS assays revealed that SeMeCys–DPGLy reduced HNSCC cell viability and proliferation at higher doses and long incubation times.
SARS-CoV-2 Spike (S) glycoprotein expression, purification and characterization in suspension human embryonic kidney cells 293
2021 - FREIRE, RENAN P.; SILVA, FELIPE D.; VITALE, PHELIPE; TODESCHINI, IRIS; MENEZES, FILIPE; OLIVEIRA, JOAO E. de; OLIVEIRA, MONA das N.; SOARES, CARLOS R.J.
SARS-CoV-2 is a zoonotic virus RNA positive, which became responsible to be the largest sanitary crisis faced by humanity: Coronavirus disease 2019 (COVID-19). Some symptoms include major sneeze conditions, who could evolve to severe acute respiratory syndrome, and in some cases, to death. Techniques for accurate detection of this virus are essential to promote an accurate diagnosis of infected patients. SARS-CoV-2 has several targets with clinical interest; although, the focuses is on Spike (S), a homotrimer glycoprotein, that interacts with angiotensin converting enzyme receptor (ACE2), developing the infection in host cells. Thus, we recognize that the demand for the glycoprotein S is necessary, requiring large amounts with high purity level. The current work has the main objective the transient expression of SARS-CoV-2 S protein into suspension human embryonic kidney cells 293 (HEK293), purification and characterization, to use it as a template for discovering new molecular markers. SARSCoV- 2 S modified protein cDNA was inserted into pαH plasmid, amplified, and purified. For transient recombinant protein expression, 7.5 x 107 HEK293 cells (Expi293FTM cells) was seeded in 27 mL Expi293™ culture Medium. The transfection was carried out with a cationic lipid ExpiFectamine™ and 30 μg of plasmid, mixed with 3 mL Opti-Mem® culture medium. Cell culture was maintained for seven days in 125 mL vent cap Erlenmeyer, 32 ºC, 8% CO2, under 125 rpm orbital shaker rotation. 10 mL aliquots were collected on four- and seven-day post transfection, stored at -80 ºC. Physical-chemical and biological characterizations were determined by SDS-PAGE, ELISA, and Western Blotting. Purification from 40 mL of conditioned medium was carried out in two steps: Strep-Tag affinity column, followed by a size exclusion Superose® 6 (10/300), 5.0 mg of oligomeric recombinant protein with 95% purity was obtained. We believe that this process can be easily adapted to different volumes, being very useful for obtaining, in a short time, enough pure and immunological active SARS-CoV-2 S for further studies and applications, such as, cryogenic electron microscopy, mass spectroscopy, N-glycan structures, antibody production and immunologic assays development.
Biophysical properties of electrospun chitosan-grafted poly(lactic acid) nanofibrous scaffolds loaded with chondroitin sulfate and silver nanoparticles
2022 - JUNIOR, ALEXANDRE F.; RIBEIRO, CHARLENE A.; LEYVA, MARIA E.; MARQUES, PAULO S.; SOARES, CARLOS R.J.; QUEIROZ, ALVARO A.A. de
The aim of this work was to study the biophysical properties of the chitosan-grafted poly(lactic acid) (CH-g-PLA) nanofibers loaded with silver nanoparticles (AgNPs) and chondroitin-4-sulfate (C4S). The electrospun CH-g-PLA:AgNP:C4S nanofibers were manufactured using the electrospinning technique. The microstructure of the CH-g-PLA:AgNP:C4S nanofibers was investigated by proton nuclear magnetic resonance (1H-NMR), scanning electron microscopy (SEM), UV-Visible spectroscopy (UV-Vis), X-ray diffraction (XRD), and Fourier transform infrared (ATR-FTIR) spectroscopy. ATR-FTIR and 1H-NMR confirm the CH grafting successfully by PLA with a substitution degree of 33.4%. The SEM measurement results indicated apparently smooth nanofibers having a diameter range of 340 ± 18 nm with porosity of 89 ± 3.08% and an average pore area of 0.27 μm2. UV-Vis and XRD suggest that silver nanoparticles with the size distribution of 30 nm were successfully incorporated into the electrospun nanofibers. The water contact angle of 12.8 ± 2.7° reveals the hydrophilic nature of the CH-g-PLA:AgNP:C4S nanofibers has been improved by C4S. The electrospun CH-g-PLA:AgNP:C4S nanofibers are found to release ions Ag+ at a concentration level capable of rendering an antimicrobial efficacy. Gram-positive bacteria (S.aureus) were more sensitive to CH-g-PLA:AgNP:C4S than Gram-negative bacteria (E. coli). The electrospun CH-g-PLA:AgNP:C4S nanofibers exhibited no cytotoxicity to the L-929 fibroblast cells, suggesting cytocompatibility. Fluorescence microscopy demonstrated that C4S promotes the adhesion and proliferation of fibroblast cells onto electrospun CH-g-PLA:AgNP:C4S nanofibers.
Microfluidic caging lipase in hyperbranched polyglycerol microcapsules for extracorporeal treatment of enzyme pancreatic insufficiency
2021 - ALVES, ANDRESSA A.; QUEIROZ, ALFREDO A.A.E. de; SOARES, CARLOS R.J.; QUEIROZ, ALVARO A.A. de
Lipase cartridges are currently the mainstay of treatment to improve fat absorption related to pancreatic insufficiency (PI) in patients receiving enteral nutrition feedings. Enzyme immobilization is an essential prerequisite for designing lipase cartridges systems for efficient enzymatic fat hydrolysis. A microfluidic approach has been adopted to produce lipase (LIP) caged in hyperbranched polyglycerol microcapsules (HPGly). The resulting HPGly-LIP microcapsules are spherical and had an average diameter of 29 µm with monomodal size distribution. The optimum conditions determined by artificial neural networks were HPGly concentration of 10 wt.%, LIP loading of 20% (wt) and total flow rate in microfluidic cell of 1.0 mL/h. Under these conditions, the maximum capacity of the LIP that can be microencapsulated is around 85% with respect to the HPGly concentration of 10 wt.% and total flow rate in microfluidic cell of 1.0 mL/h. This resultant HPGly-LIP exhibited Michaelis–Menten coefficients of 1.138,14 mM (Km) and 0.49 U/mg (Vmax) showing higher activity compared to free LIP. Finally, the robust HPGly-LIP microcapsules showed excellent recyclability. The in vitro Analysis of the HPGly-LIP cytotoxicity showed that microcapsules had no cytotoxic effect to L929 fibroblasts cells and behaved very similar to the negative control. These features will be useful for the facile construction of biocatalytic systems with high efficiency, excellent recyclability and adequate biocompatibility for treatment of patients with PI receiving enteral nutrition feedings.
Periplasmic synthesis and purification of the human prolactin antagonist Δ1‑11‑G129R‑hPRL
2021 - SUZUKI, MIRIAM F.; ALMEIDA, LARISSA A.; POMIN, STEPHANIE A.; SILVA, FELIPE D.; FREIRE, RENAN P.; OLIVEIRA, JOAO E.; AFFONSO, REGINA; SOARES, CARLOS R.J.; BARTOLINI, PAOLO
The human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.
Avaliação do cultivo de células CHO adaptadas a suspensão em diferentes formulações de meios de cultura comerciais livres de soro
2020 - ALMEIDA, LARISSA A.; SOARES, CARLOS R.J.
GH controls glycemia and metabolic adaptations to starvation via neurons that express the leptin receptor
2017 - DONATO JUNIOR, JOSE; FURIGO, ISADORA C.; OREFICE, GABRIEL; RAMOS-LOBO, ANGELA M.; SOARES, CARLOS R.J.; LIST, EDWARD O.; KOPCHICK, JOHN J.
Growth hormone (GH) responsive neurons are extensively distributed in many hypothalamic nuclei that also have leptin receptor (LepR)-expressing cells (1). However, whether GH affects metabolic functions regulated by leptin remains unknown. In the present study, we initially performed a co-localization study and confirmed that a large percentage of LepR-expressing neurons are directly responsive to peripherally injected GH in different brain nuclei. Then, we generated mice lacking GH receptor (GHR) specifically in LepR-expressing cells (LepR GHR KO mice). Although LepR GHR KO mice exhibited a similar body weight, food intake, energy expenditure, glucose tolerance and leptin sensitivity compared to control mice, we observed a lower adiposity in mutant mice. LepR GHR KO mice also showed a lower capacity to recover from insulin-induced hypoglycemia and a blunted counterregulatory response evoked by 2-deoxyglucose (2DG) administration. Co-infusion of 2DG with sympathetic blockers, but not parasympathetic blockers, was able to abolish the differences observed between groups. Remarkably, while control mice adapted to a 60% food deprivation period by progressively saving energy, LepR GHR KO mice exhibited a blunted metabolic adaptation to starvation, which led to hypoglycemia and an increased lethality rate, energy expenditure and weight loss, compared to control animals. In order to identify the specific neuronal populations responsible for the observed responses, we generated mice lacking GHR in steroidogenic factor-1 (SF1) cells, which comprises the ventromedial nucleus of the hypothalamus (VMH). SF1 GHR KO mice exhibited a similar metabolic phenotype in the basal condition, compared to littermate controls. On the other hand, SF1 GHR KO mice also showed a lower capacity to recover from insulin-induced hypoglycemia and a blunted counterregulatory response evoked by 2DG. However, metabolic adaptations to starvation were not affected by SF1-specific GHR deletion, which suggests that VMH does not mediate these latter changes. In summary, GHR expression in the brain is required to properly regulate glycemia and energy balance, especially during situations in which GH is highly secreted (e.g., hypoglycemia and food restriction). In addition, our findings revealed a previously unrecognized role of GH to coordinate, together with leptin, the metabolic adaptations to starvation in order to ensure survival, via the same neurocircuitry.