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Made available in DSpace on 2020-12-12T02:08:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-01-01 This manuscript describes the results of a metalloproteomic study of mercury in samples of muscle and liver tissue of the species Serrasalmus rhombeus, popularly known as black piranha and characterised as the most voracious and aggressive predator in the Brazilian Amazon. The metalloproteomic study involved using two-dimensional electrophoresis (2D PAGE) to fractionate the proteome of the muscle and liver tissue samples, along with atomic absorption spectrometry in a graphite furnace (GFAAS) to identify mercury associated with protein SPOTs and mass spectrometry with electrospray ionisation (ESI-MS/MS) to characterise the mercury-binding proteins. The protein SPOTs characterised showed concentrations in the order of 156 mg kg−1, which ranks as the highest concentrations of mercury determined so far in metalloproteomic studies involving fish species in the Amazon region. Based on FASTA sequences of proteins characterised by ESI-MS/MS, bioinformatics studies were performed that allowed identifying nine proteins with characteristics of biomarkers of mercury exposure. Of those proteins, glutathione peroxidase stands out as an enzyme of great importance in the antioxidant defence of organisms subjected to oxidative stress caused by xenobiotics. School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), Rua Prof R. Dr. Valter Maurício Corrêa, s/n Venom and Venomous Animals Study Center (CEVAP) of São Paulo State University (UNESP), Rua José Barbosa de Barros, 1780 - Vila Ema Institute of Biosciences São Paulo State University (UNESP), Rua Prof. Dr. Antonio Celso Wagner Zanin, 250 Faculty of Science and Engineering São Paulo State University (UNESP), R. Domingos da Costa Lopes, 780 - Jardim Itaipu School of Veterinary Medicine and Animal Science São Paulo State University (UNESP), Rua Prof R. Dr. Valter Maurício Corrêa, s/n Venom and Venomous Animals Study Center (CEVAP) of São Paulo State University (UNESP), Rua José Barbosa de Barros, 1780 - Vila Ema Institute of Biosciences São Paulo State University (UNESP), Rua Prof. Dr. Antonio Celso Wagner Zanin, 250 Faculty of Science and Engineering São Paulo State University (UNESP), R. Domingos da Costa Lopes, 780 - Jardim Itaipu

Safety, optimal minimum dose, and, preliminary effectiveness of a new generation Africanized honeybees (Apis mellifera) antivenom (AAV) were evaluated. A phase I/II, multicenter, non- randomized, single-arm clinical trial involving 20 participants showing multiple stings were studied. Participants have received either 2 to 10 vials of AAV based on the stings number together with a predefined adjuvant, symptomatic, and complementary treatment schedule. The primary safety endpoint was the presence of early adverse reactions within the first 24 hours after treatment. Preliminary efficacy through clinical evolution, including laboratory tests, was assessed at baseline and over the following four weeks. ELISA assays and mass spectrometry estimated the venom pharmacokinetics before, during, and after treatment. Twenty adult participants, 13 (65%) males, and 7 (35%) females, with a median age of 44 years and a mean body surface of 1.92 m2 (median = 1.93 m2) were recruited. The median number of stings was 52.5 ranging from 7 to more than 2,000. Envenoming severity was classified as 80% mild, 15% moderate, and 5% severe. According to the protocol, 16 (80%) participants received two AAV vials, 3 (15%) six vials, and one (5%) 10 vials. There was no discontinuation of the treatment due to acute adverse events and there were no late adverse reactions. Two patients showed mild adverse events with only transient itchy skin and erythroderma. All participants completed the infusion within two hours and there was no loss of follow-up after discharge. ELISA assays showed venom concentrations varying between 0.25 ng/mL and 1.479 ng/mL prior to treatment. Venom levels decreased in all cases during the hospitalization period. Surprisingly, in nine cases (45%), despite clinical recovery and without symptoms, the venom levels increased again during outpatient care 10 days after discharge. Mass spectrometry showed melittin in eight participants 30 days after the treatment. Considering the promising safety results of the investigational product for the treatment of massive Africanized honeybee attacks, added to efficacy in clinical improvement and immediate decrease in blood venom level, the AAV has shown to be safe for human use.Trial registrationUniversal Trial Number (UTN): U1111-1160-7011, Register Number: RBR-3fthf8 (http://www.ensaiosclinicos.gov.br/rg/RBR-3fthf8/).

Made available in DSpace on 2019-10-06T17:11:40Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 The development of metallomics techniques has allowed for metallomics analysis of biological systems, enabling a better understanding of the response mechanisms for different stimuli, their relationship to metallic species, and the characterization of biomarkers. In this study, a metallomics analysis of the muscle tissue of Nile tilapia was used to aid the understanding of the molecular mechanisms involved in zinc absorption in this fish species when fed organic and/or inorganic sources of zinc and to identify possible biomarkers for the absorption of this micromineral. To accomplish this, the fish were separated into three groups of 24 g, 74 g, and 85 g initial weights, and each group, respectively, was fed a zinc-free diet (control group, G1), a diet containing zinc found in organic sources (treatment 1, G2), and a diet containing zinc from an inorganic source (treatment 2, G3). Two-dimensional polyacrylamide (2D PAGE) gel electrophoresis was used to separate the proteins of the muscle tissue. Subsequently, the expression profiles of protein spots in the samples where zinc was applied in different concentrations were compared, using the software ImageMaster 2D Platinum version 7.0, to identify proteins that were differentially expressed. The identified proteins were then exposed to atomic absorption spectrometry in a graphite furnace to determine zinc mapping and were subsequently characterized via electrospray ionization tandem mass spectrometry (ESI-MS/MS). The metallomic analysis identified 15 proteins differentially expressed and associated with zinc, leading to the conclusion that three metal-binding proteins presented as possible biomarkers of zinc absorption in fish. School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP) Federal University of Mato Grosso do Sul (UFMS) Biochemistry Department São Paulo University (USP) Biochemistry Department University of Nebraska-UNL School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) Institute of Biosciences São Paulo State University (UNESP)

Made available in DSpace on 2018-12-11T16:47:41Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-12-01 Bioaccumulative metals such as mercury are found in increasing amounts in fish and their consumers. In the region of the Madeira River, in the Brazilian Amazon, mercury (Hg) is a predominant contaminant in the aquatic ecosystem. There is therefore a need to find specific biomarkers of mercury toxicity in fish to monitor contaminations. Here, mercury-bound proteins were identified in the liver tissues of fishes Mylossoma duriventre and Brachyplatystoma rousseauxii. Mercury was quantified in liver tissue, pellets and protein spots by graphite furnace atomic absorption. Proteins were fractionated by two-dimensional polyacrylamide gel electrophoresis and identified by mass spectrometry with electrospray ionization. We identified nine proteins linked to mercury and that presented biomarker characteristics of mercury. Among the proteins identified, isoforms of parvalbumin, ubiquitin-40S ribosomal protein S27a, brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 2 and betaine–homocysteine S-methyltransferase 1 are notable for having the molecular function of binding to metallic ions. Institute of Biosciences São Paulo State University (UNESP) University of Nebraska-Lincoln School of Veterinary Medicine and Animal Science São Paulo State University (UNESP) University of São Paulo (USP) College of Planaltina University of Brasília (UnB) Institute of Biosciences São Paulo State University (UNESP) School of Veterinary Medicine and Animal Science São Paulo State University (UNESP)

Made available in DSpace on 2020-12-10T19:43:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-03-20 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Sustainable Energy P&D project of Brazil S.A. The high concentrations of mercury found in Amazon have been intensively studied by the scientific community in the last decades. These mercurial species bind preferentially to proteins. Therefore, this work proposal sought to obtain the fractionation, identification and study of mercury - bound proteins present in samples of muscular and hepatic tissue from fish collected in the reservoir of the Jirau Hydroelectric Power Plant - on the Madeira River. Two-dimensional electrophoresis (2D-PAGE) for protein fractionation, graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of mercury and Mass Spectrometry (ESI-MS/MS) were used for the identification of proteins. Concluding the work with analysis of graphics from the Blast2go program. Two mercury - bound proteins were identified as triosephosphate isomerase A and Protein FAM45A. The data generated by the bioinformatics programs confirm the tendency of these proteins to be linked to mercury and elucity the possibles existing physiological and cellular interactions. Sao Paulo State Univ Julio de Mesquita Filho, Dept Chem & Biochem, Botucatu, SP, Brazil Univ Sao Paulo, Bauru Fac Dent, Sao Paulo, SP, Brazil Univ Brasilia, Brasilia, DF, Brazil Sao Paulo State Univ Julio de Mesquita Filho, Dept Chem & Biochem, Botucatu, SP, Brazil FAPESP: 2014/02638-5 FAPESP: 2013/21297-1 FAPESP: 2016/19404-2 CNPq: 304768/2018-9 CNPq: 404485/2016-2 Sustainable Energy P&D project of Brazil S.A.: 6631-0001/2012 Sustainable Energy P&D project of Brazil S.A.: 004/13

This study used metalloproteomic techniques to characterize mercury (Hg)-bound proteins in the muscle and liver tissue of Tucunare (Cichla spp.) collected at the Jirau Hydroelectric Power Plant in Madeira River Basin, Brazil. The proteome of the muscle and liver tissue was obtained after two steps of fractional precipitation and separating the proteins by 2-D polyacrylamide gel electrophoresis. Hg was identified and quantified in the protein spots by graphite furnace atomic absorption spectrometry after acid mineralization in an ultrasound bath. Hg with a molecular weight

Submitted by Bruna Cavecci Mendonça (brucavecci@gmail.com) on 2017-04-19T17:38:01Z No. of bitstreams: 1 TESE Bruna.docx: 8277927 bytes, checksum: c7e5757153327028ae2d15466a1ef476 (MD5) Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: A versão final da dissertação/tese deve ser submetida no formato PDF (Portable Document Format). O arquivo PDF não deve estar protegido e a dissertação/tese deve estar em um único arquivo, inclusive os apêndices e anexos, se houver. Por favor, corrija o formato do arquivo e realize uma nova submissão. Agradecemos a compreensão. on 2017-04-19T19:48:09Z (GMT) Submitted by Bruna Cavecci Mendonça (brucavecci@gmail.com) on 2017-04-19T19:56:04Z No. of bitstreams: 1 TESE Bruna.pdf: 4516699 bytes, checksum: 52d15f9321a7769728ef47dc8f836b95 (MD5) Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-19T20:12:56Z (GMT) No. of bitstreams: 1 mendonca_bc_dr_bot_par.pdf: 970963 bytes, checksum: f51b437518719dfb68d1cdb6edf71335 (MD5) Made available in DSpace on 2017-04-19T20:12:56Z (GMT). No. of bitstreams: 1 mendonca_bc_dr_bot_par.pdf: 970963 bytes, checksum: f51b437518719dfb68d1cdb6edf71335 (MD5) Previous issue date: 2017-02-23 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Outra O peixe é uma importante fonte de proteínas de alto valor biológico, vitaminas e minerais. Porém, este alimento é também uma das principais vias de exposição humana a contaminantes tóxicos como o metilmercúrio (MeHg). Assim a sua acumulação no peixe pode predispor sérios riscos para a saúde humana. As altas concentrações de mercúrio encontrados em solo, sedimentos, peixes e humanos na Amazônia brasileira têm sido estudados intensamente pela comunidade científica nas últimas décadas, procurando-se elucidar os mecanismos de toxicidade das espécies mercuriais. As espécies mercuriais ligam-se preferencialmente às proteínas. Podendo significar que a fração biodisponível das espécies mercuriais nas diferentes espécies de peixes pode ser dependente da forma como estas estão ligadas a essas proteínas. Portanto esta proposta de trabalho buscou otimizar procedimentos eletroforéticos por 2D-PAGE para fracionamento e identificação de proteínas responsáveis pelo transporte de mercúrio em amostras de tecido muscular e hepático de peixes coletados no reservatório da Usina Hidrelétrica de Jirau – no rio Madeira; utilizando o método de espectrometria de absorção atômica em forno de grafite (GFAAS), para determinação de mercúrio nos tecidos e spots proteicos obtidos; avaliou o estresse oxidativo dos peixes contaminados com mercúrio por meio das atividades das enzimas catalase (CAT), glutationa peroxidase (GSH-Px) e superóxido dismutase (SOD), bem como das concentrações de hidroperóxido de lipídio (HP); e caracterizou as proteínas transportadoras de mercúrio por Espectrometria de Massas (ESI-MS/MS) e posteriormente por Shotgun que envolve a união de duas técnicas a Espectrometria de Massas e a Cromatografia Líquida de Alta Eficiência (ESI-MS/MS + HPLC), finalizando o trabalho com ferramentas de bioinformática. Nesse trabalho três espécies de peixes, apreciadas na região e também comercializadas, foram estudadas Brachyplatystoma rousseauxii (Dourada), Mylossoma sp., Myleus sp. (Pacu) e Prochilodus nigricans (Curimatã), utilizou-se o tecido muscular e hepático de cada uma dessas espécies. Os resultados obtidos demonstram alta concentração de mercúrio no tecido hepático das três espécies de peixe estudadas numa média treze vezes mais concentrada do que no tecido muscular. Após a otimização de extrações, precipitações e corridas eletroforéticas, foram identificadas duas proteínas ligadas ao mercúrio a Triosephosphate isomerase A em seis spots proteicos sendo 5 deles no fígado de Dourada e 1 spot proteico no fígado de Pacu, e a Protein FAM45A no spot proteico de fígado de Cutimatã. Os dados gerados pelos programas de bioinformática confirmam a tendência dessas proteínas estarem ligadas ao mercúrio. Fish is an important source of high biological value proteins, vitamins and minerals. However, this food is also one of the main routes of human exposure to toxic contaminants such as methylmercury (MeHg). Thus, their accumulation in the fish may predispose serious risks to human health. The high concentrations of mercury found in soil, sediments, fish and humans in the Brazilian Amazon; have been studied intensively by the scientific community in the last decades, trying to elucidate the mechanisms of mercury toxicity. Mercurial species bind preferentially to proteins. It may mean that the bioavailable fraction of mercury species in different species of fish may be dependent on the way they are bound to these proteins. Therefore, this work proposal sought to optimize 2D-PAGE electrophoretic procedures for fractionation and identification of proteins responsible for the transport of mercury in samples of muscle and liver tissue collected in the reservoir of the Jirau Hydroelectric Power Plant - on the Madeira river; using atomic absorption spectrometry in graphite furnace (GFAAS), for the determination of mercury in the tissues and protein spots obtained; Evaluated the oxidative stress of fish contaminated with mercury through the activities of the enzymes catalase (CAT), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), as well as the concentrations of lipid hydroperoxide (HP); and characterized by mercury transport proteins by Mass Spectrometry (ESI-MS/MS) and later by Shotgun involving the combination of Mass Spectrometry and High Performance Liquid Chromatography (ESI-MS/MS + HPLC) Finalizing the work with bioinformatics tools. In this work three species of fish, appreciated in the region and also commercialized, were studied Brachyplatystoma rousseauxii (Dourada), Mylossoma sp., Myleus sp. (Pacu) and Prochilodus nigricans (Curimatã), the muscle and liver tissue of each of these species was used. The results obtained demonstrate a high concentration of mercury in the hepatic tissue of the three fish species studied in an average thirteen times more concentrated than in muscle tissue. After optimization of extractions, precipitations and electrophoretic runs, two mercury-bound proteins were identified: Triosephosphate isomerase A in six protein spots, five of them in the liver of Dourada and one protein spot in the liver of Pacu, and Protein FAM45A in the protein spot of Cutimatã liver. The data generated by the bioinformatics programs confirm the tendency of these proteins to be linked to mercury. FAPESP: 14/02638-5

Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-27T11:29:47Z No. of bitstreams: 0 Made available in DSpace on 2014-05-27T11:29:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-06-27 In the present study, a simple, rapid and sensitive method was developed for the determination of mercury concentrations in the muscle tissue of fish from the Brazilian Amazon using graphite furnace atomic absorption spectrometry (GFAAS) following acid mineralization of the samples in an ultrasonic cold water bath. Using copper nitrate as a chemical modifier in solution and sodium tungstate as permanent modifier, we were able to attain thermal stabilization of the mercury up to the atomisation temperature of 1600 °C in the GFAAS assay. The calculated limits of detection (LOD) and quantification (LOQ) were 0.014 and 0.047 mg kg-1, respectively. © 2013 Elsevier Ltd. All rights reserved. UNESP - São Paulo State University Biosciences Institute, Botucatu, SP UNESP - São Paulo State University Faculty of Veterinary Medicine and Animal Science, Botucatu, SP University of Alagoas - IQB/UFAL, Maceió, Al University of Brasília Faculdade de Planaltina Distrito Federal National Institute of Bioanalytical Sciences and Technology UNICAMP - Campinas State University, Campinas, SP UNESP - São Paulo State University Biosciences Institute, Botucatu, SP UNESP - São Paulo State University Faculty of Veterinary Medicine and Animal Science, Botucatu, SP

This paper presents the results obtained from selenium fractionation in plasma, muscle and liver samples of Nile tilapia’s (Oreochromis niloticus) after protein separation. The plasma, muscle and liver proteome was obtained by 2D-PAGE, and selenium in protein spots was qualitatively and quantitatively determined by synchrotron radiation X-ray fluorescence and graphite furnace atomic absorption spectrometry (GFAAS). The fluorescence spectra indicated the presence of selenium in three protein spots of plasma, two of muscle and one of liver. Selenium was found to be distributed mainly in proteins with a molar mass smaller than 57.0 kDa and with pI in the range of 5.9–9.6, with one exception in the plasma sample, which presented protein with a molar mass of 60.0 kDa. After acid mineralization of the protein spots, a GFAAS determination of the concentration of selenium bound to these proteins indicated a range of 1.35–6.82 mg per g of protein.

Made available in DSpace on 2018-12-11T17:00:50Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-03-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The aim of this paper was to determine the zinc concentrations in muscle samples of Nile tilapia (Oreochromis niloticus) produced in aquaculture and fed diets containing inorganic and organic zinc sources. In the sample preparation step, the samples were lyophilized and ground cryogenically to produce particles smaller than 60 µm. The zinc extraction was conducted by acid mineralization using concentrated nitric acid and hydrogen peroxide in a thermostatically controlled ultrasonic batch reactor. The zinc determinations were performed by Graphite Furnace Atomic Absorption Spectrometry at a drying temperature of 90–250 °C, pyrolysis temperature of 1400 °C, atomization temperature of 2400 °C, and cleaning temperature of 2800 °C. Niobium nitrate was used as a chemical modifier co-injected with the samples, and tungsten was employed as a permanent modifier. Among the muscle tissue samples of Nile tilapia that were analyzed, zinc concentrations were determined in the range of 3.70–19.00 mg kg−1 (LOD 0.012 µg L−1 and LOQ 0.041 µg L−1). The accuracy and precision of the proposed method of zinc determination was validated using the DORM-certified standard Fish Protein 4 NRC. College of Veterinary and Animal Science São Paulo State University Department of Chemistry and Biochemistry Bioscience Institute São Paulo State University (UNESP), Rubião Júnior Federal University of Alagoas - Institute of Chemistry and Biotechnology College of Veterinary and Animal Science São Paulo State University Department of Chemistry and Biochemistry Bioscience Institute São Paulo State University (UNESP), Rubião Júnior FAPESP: 2010/51332-5 FAPESP: 2010/51562-0

Made available in DSpace on 2018-12-11T16:58:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The feeds supplied to farmed fish should be rich in protein and minerals, such as animal protein, but more expensive alternatives comprising vegetable protein have emerged in recent years. However, the minerals essential to the proper development of fish must be assessed. To determine whether the minerals supplied in the diet are being fully exploited, the equation of the apparent digestibility coefficient is typically used with an external marker such as chromium. Moreover, the concentrations of the metals of interest are usually obtained using an acid digestion in the digester block; however, this digestion is harmful to both the analyst and the environment. An alternative to this digestion is described in this work. Four types of feeds were made with different types of protein substitutes. Department of Chemistry and Biochemistry Institute of Bioscience São Paulo State University (UNESP), Rubião Junior District College of Veterinary and Animal Science São Paulo State University Department of Chemistry and Biochemistry Institute of Bioscience São Paulo State University (UNESP), Rubião Junior District College of Veterinary and Animal Science São Paulo State University FAPESP: 2008/51377-9

Made available in DSpace on 2015-10-21T13:11:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-01. Added 1 bitstream(s) on 2015-10-22T09:52:13Z : No. of bitstreams: 1 S0103-50532015000200239.pdf: 519548 bytes, checksum: bd9cc7af88b59229a1869b3e0cfb4781 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study evaluated the metalloproteomic profile of muscle tissue samples from Nile tilapia (Oreochromis niloticus). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods was used for protein fractionation and identification based on image analysis. Determination of Ca, Cu, Fe, Mn and Zn was performed in the protein spots by flame and/or graphite furnace atomic absorption spectrometry (FAAS, GFAAS), the protein spots were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). FAAS and GFAAS determinations have indicated the presence of calcium in seven protein spots, iron in only one spot, manganese in six spots, and zinc in two spots. Of the seventeen protein spots where the presence of metal ions was detected, ten were characterized by ESI-MS/MS. UNESP, Coll Vet &Anim Sci, BR-18618970 Botucatu, SP, Brazil UNESP, Inst Biosci, Dept Chem &Biochem, BR-18618970 Botucatu, SP, Brazil UNESP, Dept Biol Sci, BR-18618970 Botucatu, SP, Brazil UNESP, Coll Vet &Anim Sci, BR-18618970 Botucatu, SP, Brazil UNESP, Inst Biosci, Dept Chem &Biochem, BR-18618970 Botucatu, SP, Brazil UNESP, Dept Biol Sci, BR-18618970 Botucatu, SP, Brazil FAPESP: 2011/14902-0 FAPESP: 2010/51332-5

This study aimed to identify metalloproteins that lose their metal ions in the presence of mercury (Hg) and bind to Hg in breast milk samples collected from the riverine population of the Madeira River, a tributary of the Amazon River. Initially, total Hg was determined from the hair of lactating women to identify individuals who were contaminated followed by a proteomic analysis of breast milk samples through two-dimensional polyacrylamide gel electrophoresis after acetone precipitation. The presence of Hg in the obtained protein spots was determined by cold vapor atomic absorption spectrometry and cold vapor atomic fluorescence spectrometry. These determinations indicated the presence of Hg in one protein spot, which was then characterized through electrospray ionization tandem mass spectrometry. Based on searches in the UniProt database, this protein spot was identified as lysozyme C.