PPG - Bioprodutos e Bioprocessos

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Alterações em conchas de Lottia Subrugosa como possível biomarcador de contaminação para áreas costeiras multi-impactados
(Universidade Federal de São Paulo, 2018-06-29) Santos, Nayara Gouveia dos [UNIFESP]; Castro, Ítalo Braga [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Molluscs Have Been Widely Used In Biomonitoring Of Aquatic Environments Because They Are Highly Sensitive To The Effects Of Hazardous Chemicals And Have The Capacity To Store In Shells The History Of Environmental Changes In Their Habitat. Lottia Subrugosa Is A Patelliform Gastropod Specie Found In Tidal Zones From Brazilian Coast. This Organism Is A Promising Tool To Be Used In Environmental Assessments In Monitoring Studies. The Present Study Evaluated The Association Among Traditional Biochemical Biomarkers With Biometric, Morphometric, And Elemental Composition Of L. Subrugosa Shells From Three Multi-Impacted Coastal Areas In Brazil. The Study Was Carried Out In Todos Os Santos Bay (Tsb), Santos/São Vicente Estuarine System (Sses) And Paranaguá Estuarine Complex (Pec), Using Three Sampling Sites To Seek Contamination Gradients On Each Area. Results Showed That All Biomarkers Evaluated Responded To Environmental Contamination, Regardless The Presence (Sess And Cep) Or Absence (Tsb) Of A Gradient Of Contami
Acesso aberto (Open Access)
Bioprospecção alvo-direcionada de moduladores da família das proteínas inibidoras de apoptose (IAPS) em extratos de microrganismos marinhos
(Universidade Federal de São Paulo, 2018-09-21) Barbosa, Gabriel Henrique [UNIFESP]; Jimenez, Paula Christine [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Introduction: Electing a good molecular target is still a hindrance regarding the discovery of new drugs. The Inhibitor of Apoptosis Protein family (IAPs) is a relevant target in cancer research, as we find members of this class overexpressed in several tumor types and almost totally absent in normal cells. Objective: Thus, this work aimed to prospect, through a target-guided approach, natural products produced by marine actinobacteria that act as modulators of the IAP family, validating its activity and interaction through in vitro assay. Methods: Three members of the IAP family (i.e. survivin, livin and XIAP) obtained by heterologous expression in E. coli vector were attached to a resin support and incubated with marine actinobacteria extracts using a bioaffinity assay called functional chromatography. Molecules retained in the resin were eluted and analyzed by HPLC-MS, resulting directly in the mass of the ligand. We fractionated the crude extract of the selected actinobacteria (BRA-214) in Sephadex-LH20, silica cartridge, semi-preparative HPLC and manually collected in analytical HPLC, monitored by DAD detector. Isolated compounds were tested for their IC50 in SK-MEL-19 cells and, by Western blotting, inquired about their effects on protein expression of the IAP survivin and other relevant apoptosis markers. Results: 19 crude extracts were submitted to bioaffinity assays; two of them resulted in hits. The selected extract, produced by actinobacteria BRA-214, previously identified as a member of the Streptomyces genus, returned hits of m/z 613, 670, 684, 704, 718 and 752 (M+H+]. Four of them (m/z 613, 704, 718 and 752) were successfully isolated and seem to be unpublished structures. Their fragmentation profile indicates that they belong to the same class of non-ribosomal peptides, except for the 613 m/z hit, which seems to be from another unknown class. The treatment with the compound of m/z 704 promoted a lower expression of survivin in SK-MEL-19 cells. Conclusion: Functional chromatography showed to be a valid and applicable technique to bioprospect ligands to protein from the IAP family, allowing the identification and recovery of unprecedented secondary metabolites that seem to interact and modulate this family of proteins.
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Caracterização físico-química e avaliação do efeito biológico da biosilica proveniente da espécie de esponja marinha tedania ignis
(Universidade Federal de São Paulo, 2018-09-21) Cruz, Matheus de Almeida [UNIFESP]; Renno, Ana Claudia Muniz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Bone fractures are an important event in the medical clinic and are mainly related to the occurrence of trauma, accidents, sports injuries and diseases. In this context, biomaterials have been gaining prominence as an alternative in the treatment of fractures, especially the active bioglass 45S5, which is considered the gold standard of performance in the treatment of fractures. However, the great problem of this resource is due to its high cost of manufacturing or importation, which makes its access to the population restricted. Thus, research that seeks to prospect for natural products that have a good performance in bone repair becomes more and more necessary. In this context, marine sponges, which are already the marine organisms most used in medical research, present in their skeleton an organic portion, which is an analogue of collagen type XIII called spongin, in addition to an inorganic portion called Biosilica (BS), which makes up the spicules of these marine organisms. In this context, the objective of this work was: (i) to characterize physicochemically the BS and (ii) evaluate the biological effect of BS by in vivo analyzes. BG 45S5 was used for comparison in both cases. The characterization was done through MEV / EDX, XRD, FTIR, Calcium test and pH. For the in vivo tests, histopathological, histomorphometric and three-point biomechanical tests were performed. The results of SEM / EDX indicated that BS, after its extraction, showed spike structures with presence of Si (Silica) and O (oxygen) elements in its composition, while BG presented particles of varying size and shape with presence of the elements Si (Silica), O (Oxygen), Ca (Calcium), Na (Sodium), Al (Aluminum), Mg (Magnesium) and P (Phosphorus). In the FTIR results, it was possible to observe peaks referring to the groups Si-OH and Si-O-Si in BS and BG, besides the BG present a peak referring to the P-O. The XRD spectrum demonstrated the predominantly amorphous character of BS, but still with crystalline peaks characteristic of silica containing samples, while BG was completely amorphous. In the Ca assay, BS on day 1 demonstrated mineralization while BG demonstrated ion release from the incubation solution. However, from day 3, BS and BG demonstrated to release Ca in the incubator solution, this process being more prominent in BG until the last period. The pH analyzes allowed to observe that BS acidified the incubation medium on day 1 and, from the third day, the same reached a plateau near the physiological pH, while BG demonstrated to alkalinize the medium of day 1 until day 21. The results of the histopathological analysis, in tibiae of rats after 15 days, demonstrated extensive formation of bone tissue in Control Group (GC), presenting areas of osteoid tissue. BS demonstrated to be biocompatible, with initial formation of neoformed bone tissue, mainly in the periphery of the defect, presenting osteoid tissue, besides extensive formation of granulation tissue organized around BS particles. BG presented areas of formation of neoformed bone tissue largely than BS, with a large volume of osteoid tissue interspersed by BG particles and less evident granulation tissue. The histomorphometric parameter for the osteoblastic surface (Ob.S / BSf) demonstrated the bioactivity and anabolic effect of Biosilica. Biomechanical analysis demonstrated good mechanical properties of BS. Finally, it was possible to conclude that BS may present great potential in the fracture repair process, however, more biological studies with longer experimental periods and with different forms of presentation of the material are still necessary, in order to elucidate the mechanisms involved in the repair and to optimize the performance of this new material.
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Bioprospecção de microorganismos recuperados do sedimento do litoral de são paulo com potencial anticâncer
(Universidade Federal de São Paulo, 2018-09-24) Rigato, Dhiego Botelho [UNIFESP]; Jimenez, Paula Christine [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The oceans have proven to be an immeasurable source of new molecules with pharmacological applications, especially among microorganisms from the diverse Actinobacteria class. The goal of this study was to bioprospect the anticancer potential of extracts produced by marine actinobacteria recovered from sediments collected on the coast of São Paulo. Sediment samples were collected across 10 sites on the coast of São Paulo and processed by two methods and three culture media. Purified strains were grown in liquid media and extracted using ethyl acetate. The 231 extracts obtained from the respective strain were analyzed for cytotoxicity against HCT-116 cells (human colorectal carcinoma) by the MTT assay at two concentrations (5 µg/mL e 50 µg/mL). Extracts that inhibited greater than 75% of cell growth in both concentration were selected for quantitative approach. Values for the IC50 for 25 extracts were obtained after 72h incubation with tree tumor cell lines (HCT-116, MM200, MCF7) and one non-tumor cell line (RPE). Two extracts (BRB-094 and BRB-104), the producers of which were identified as members of the Streptomyces genera, were then selected according to their chemical and biological profile for further studies of cell viability and morphological analysis. BRB-094 caused a significant decrease in the number of viable cells after 48h and 72h incubation at the highest concentration tested (0.5µg/mL). Regarding the number of non-viable cells, the extract induced a significant increase at both concentrations tested (0.5µg/mL and 0.1µg/mL). This result suggests that cells exposed to this extract may be undergoing a process of death. BRB-104, on the other hand, caused a significant decrease in the number of viable cells without increasing the number of non-viable cells, which may be suggestive of an effect on the reduction of cell proliferation. These results have shown that the coast of São Paulo holds a rich biotechnological potential in the marine microorganisms.
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Prospecção biotecnológica de microrganismos marinhos com potencial anticâncer recuperados de sedimento do arquipélago de alcatrazes e sua utilização como suporte à conservação
(Universidade Federal de São Paulo, 2018-09-25) Falcão, Gabriela Ciscotto [UNIFESP]; Jimenez, Paula Christine [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The Alcatrazes Archipelago (AA), after nearly 30 years of discussions, and based on research that demonstrated its ecological relevance, the presence of endemic species that were endangered and also some rare species, became a Federal Conservation Unit (CU), declared a Wildlife Refuge in August 2016. In this context, and with the growing discoveries of genes and molecules from marine species potentially endowed with therapeutic value, such as drugs, the goal of the present study was to evaluate the biotechnological patrimony housed in microorganisms recovered from the AA sediment based on the evaluation of the cytotoxic potential of their organic extracts in a model of tumor cells in culture, metabolomic analysis of these extracts and molecular identification of the strains considered most relevant. A total of 39 strains of bacteria were recovered from the sediment and, of these, 17 produced extracts that were active in the cytotoxicity tests. Through a metabolomic analysis by molecular network generated on the GNPS platform, it was possible to identify the occurrence of the molecules such as Surfactin, Colic Acid, Ferrioxamine and Desferrioxamine among the analyzed extracts, in addition to a large number of unidentified precursor ions. The 17 active strains underwent molecular identification at the genus level and a construction of a clustering tree, where it was possible to observe that among them there were bacteria of the genera Bacillus, Pseudomonas, Halomonas and Psychrobacter. There was a pattern in the presence of the molecules identified within the genus, indicated by clusters of precursor ions unique to extracts obtained from bacteria of the genus Bacillus, whereas other clusters were typical of Gram-negative bacteria and, therefore, present in the other genera elucidated. The results presented herein demonstrate the biotechnological potential housed in the AA. As a new CU, the sustainable exploration of the Alcatrazes Archipelago must have great relevance in the perspective of economically valuing species that do not have other measurable value, deriving new bioproducts with minimum impacts to the region and, thus, harness the conservation of this ecologically valuable marine area of Brazil to the provision of this important ecosystem service.
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Avaliação ecotoxicológica de sedimentos contaminados com poluentes emergentes empregando o organismo Callinectes SP
(Universidade Federal de São Paulo, 2018-09-28) Moreno, Beatriz Barbosa [UNIFESP]; Cesar, Augusto [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Population Growth In Coastal Zones Has Generated Environmental Impacts Of Various Natures, Among Which Can Be Highlight The Inadequate Disposal Of Industrial And Domestic Effluents In Marine And Estuarine Waters. Domestic Effluents Carry A Wide Variety Of Contaminants Such As Pharmaceuticals And Personal Care (Ppcps), Which Can Be Classified As Emerging Pollutants. With This, The European And North American Environmental Agencies Began To Regulate The Approval, Commercialization And Disposal Of These Pollutants Through Directives. It Has Been Shown That Changes In Biological Responses Are An Important Tool In Assessing The Risks Associated With The Introduction Of These Substances In The Environment, Since The Responses Observed Are Sensitive To The Exposure And Effect Of These Xenobiotics, Providing Relevant Information On Their Bioavailability And Toxicity. The Aim Of The Present Study Was To Determine The Environmental Concentrations Of Triclosan (Tcs) And Ibuprofen (Ibu) In Marine Sediments Around The San
Acesso aberto (Open Access)
Revisão sistemática sobre uso de resíduos de pescados para produção de biogás e bioprodutos
(Universidade Federal de São Paulo, 2018-11-18) Sousa, Luciano Moura Beserra de [UNIFESP]; Penteado, Eduardo Dellosso [UNIFESP]; http://lattes.cnpq.br/2585863255642834; https://lattes.cnpq.br/8408014032701938; Universidade Federal de São Paulo (UNIFESP)
Pescados são uma fonte de alimento humano, conhecida, comum e de consumo mais elevado em áreas costeiras. Nos últimos anos, houve um aumento no consumo dessa proteína o que automaticamente também incrementou a produção de resíduos que impactam o meio ambiente. Um dos principais impactos da indústria pesqueira, que muitas vezes desenvolve suas atividades em situações de pouca regulamentação, monitoramento, e realizando exploração predatória, é a não destinação adequada dos seus resíduos. Entretanto, muitos trabalhos têm proposto o tratamento da fração orgânica dos resíduos em biodigestores anaeróbios, visando gerar biogás que pode ser usado como fonte energética e minimizar os impactos causados. Neste sentido, esse trabalho teve como objetivo desenvolver uma revisão bibliográfica sistemática sobre a utilização de resíduos de pescados como matéria prima para produção de biogás e bioprodutos, através da digestão anaeróbica identificando a eficiência na degradação dos resíduos da indústria de pescado e os principais parâmetros operacionais adotados. Para isso, a metodologia incluiu fases de busca de artigos nos principais buscadores acadêmicos (Scopus, Pubmed, Science Direct), de triagem (publicação nos últimos 5 anos – de 2017 a 2021, em língua inglesa e que seja artigo de pesquisa original), de extração e de análise dos dados. Os artigos foram categorizados com base na metodologia PRISMA, sendo uma referência para eliminar o efeito negativo do risco de viés. Ao todo foram obtidos 17 artigos, concidentemente em sua maioria com enfoque laboratorial, sendo produzida uma análise de viés, que respalda a possibilidade de aplicação do uso de resíduos de peixe como substrato e cosubstrato para produção de biogás, ainda que pouco utilizados em escalas reais e fora do ambiente acadêmico. O biogás produzido em mais de 50% dos artigos selecionados apresentou valores médios entre 201 e 800 mL CH4 g -1 SV, a média de produção de metano com cosubstrato foi cerca de 24% maior do que sem o uso de cosubstrato (469,73 ante 379,12 ml CH4 g -1 SV, respectivamente). As melhores condições para a produção de biogás em biodigestores anaeróbios foram obtidas em temperatura mesofílica de 37 °C como predominante e teve-se o controle do pH da parte líquida durante o processo. Os resultados da pesquisa baseados nos artigos demonstram potencial de resíduos de pescados como fonte para biometano e biogás, mas com necessidade de mais aprofundamento e pesquisas que ultrapassem a dimensão laboratorial e abordem um possível uso comercial e social, incluindo abordagem socioambiental os aspectos técnicos.
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Efeitos de frações isoladas do veneno da formiga dinoponera quadriceps (kempf) em modelo de crises convulsivas induzidas por bicuculina em camundongos
(Universidade Federal de São Paulo (UNIFESP), 2019-02-25) Freitas, Francisca Rayanne Da Silva [UNIFESP]; Ribeiro, Alessandra Mussi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Natural products are recognized as therapeutic tools for treating several diseases since ancient civilizations. In this context, animal venoms and their isolated components have shown great biotechnological potential, because these substances can be neuroactive with high affinity and specificity by nervous tissue in invertebrates and vertebrates. Thus, poisons can be used as instruments that help in the understanding of neuropathological alterations that occur in the human’s brain, such as epilepsy. Epilepsy is a neurological disorder that affects millions of individuals worldwide, and despite of the therapeutic advances many patients develop pharmacoresistance. It is inequivocal the need for the development of new substances that can be used as drug-model for new antiepileptic drugs. Previous studies have shown anti- and convulsant effects of the venom of the ant Dinoponera quadriceps. The aim of this study was to isolate the fractions of the D. quadriceps venom and to evaluate the neuroprotective potential on seizures induced by bicuculine (BIC) in mice. Crude venom was fractionated by high performance liquid chromatography (HPLC) resulting in 68 fractions (referred to as DqtxII-1 to 68). Male Swiss mice (3 months) were implanted with a cannula in the lateral ventricle. After the recovery period, animals were previously microinjected with saline solution (saline, n = 11) or each fraction (n = 114), afterwards each animal was observed in an open field (20 min). For the evaluation of the neuroprotective activity, after 30 min the animals of the BIC group (saline x BIC, n = 9) and fractions (Dqtxs x BIC, n = 111) were microinjected with bicuculline (10 mg/mL) and observed in CA (20 min). Our main results showed that there was not significant difference in the exploratory, grooming and immobility behaviors in mice treated with Dqtxs. Regarding the anticonvulsive activity, DqtxII-7 treated animals showed high latency to appear of the seizures (971 ± 164 s) when compared with BIC group (252 ± 91 s, p = 0,007). Further, this fraction protected 71% and 86% of animals against seizures and death, respectively. Thus, new studies are needed to determine the its structure and mechanism of action.
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Alterações estruturais em conchas de lottia subrugosa como biomarcadores de contaminação costeira
(Universidade Federal de São Paulo (UNIFESP), 2019-03-08) Oliveira, Carolina Rocha Moreira de [UNIFESP]; Castro, Ítalo Braga [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Molluscs have been widely used in biomonitoring of aquatic environments because they are highly sensitive to hazardous chemical substances and have the capacity to express in their shells the history of environmental changes in their habitats. The shells of these organisms also have biomolecules which play an essential role in the biomineralization process. Thus, the present study evaluated aspects of the shell structure of Lottia subrugosa gastropods along the spatial gradient of contamination in the Santos estuary. The organism were caught at Ponta da Praia (S1), Praia do Góes (S2) and Palmas island (S3) representing decreasing levels of contamination. After sampling, a batch of shells from each sampling point was submitted to a compression test to quantify their resistance to mechanical forces. Afterwards, another pool of shells from the same sites was submitted to thermogravimetric analysis (TGA) to evaluate its organic fraction, followed by scanning electron microscopy (SEM) to visualize transversal structural profile. Then, the acid-soluble shell protein matrix (PMC) were extracted and quantitatively analyzed. The results showed that shells of organisms collected in more contaminated areas were more fragile requiring less amount of force to break during the compression tests. Similarly, the results obtained in the TGA analysis indicated that shells from less contaminated areas had a higher percentage of organic substances when compared to shells from impacted areas. The SEM micrographs also showed a reduction in the thickness of the prismatic layer associated with the enlargement of the periostracum layer in the most contaminated sites. In addition, significant changes in PMC concentrations were observed demonstrating a tendency to reduce the levels in more contaminated areas. This suggests that organisms exposed to contamination may be more vulnerable to predatory pressures. This trend, highly related to the results of the compression tests, suggests that the vulnerability observed in the shells of more contaminated zones is related to a possible loss of organic matrix of the shells, as well as the observed structural alterations. This relationship can be explained by the reduction in protein content, however, more tests are needed to improve the qualitative analysis of PMCs.
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Avaliação temporal da forma e composição de conchas de lottia subrugosa do litoral do estado de São Paulo
(Universidade Federal de São Paulo (UNIFESP), 2019-03-08) Martins, Camila Pratalli [UNIFESP]; Castro, Ítalo Braga [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The intense human occupation and the development of industrial and tourist activities in coastal areas have caused several environmental impacts, especially result of launching of domestic and industrial effluents without treatment in coastal waters. Alteration in the mollusc shells have been recently related to the exposure of contaminants. It is also known that the shells of these animals preserve an important history about their growth, mineralization conditions and environmental conditions in which they lived. Thus, this study carried out a temporal assessment of alterations in shape and composition of shells of the Lottia subrugosa gastropod (D'ORBIGNY, 1846) from coastal areas of the State of São Paulo. For that, an inventory of the L. subrugosa lots deposited in zoological collections, before 1985, in main Brazilian museums was carried out. Subsequently 50 living individuals were collected in the same areas of the selected lots. The shells (from the collections and obtained in the environment) were analyzed using techniques of biometry, 2D geometric morphometry and analysis of the elemental chemical composition. The results obtained by the comparison by locality, between organisms of the past and the present, did not demonstrate a clear pattern of alterations of the forms along the time. This was probably due to the restrictions imposed on the work, regarding the impossibility of determining the age of the organisms used and the lack of lots with temporal sequences coming from the study areas. On the other hand, when these same forms of organisms were analyzed together, between present and past, it was possible to perceive that the present organisms present a smaller variability of forms when compared with the group of the past. This same pattern, on the other hand, was not observed when investigating the biometric variations and the elemental composition. Considering the tools used in the present study, the geometric morphometry presented consistent answers for the quantification of differences in the shape of the shell of Lottia subrugosa in the temporal character, although it was not possible to infer direct relation with the environmental contamination. This technique has been shown to be a promising tool, relatively simple, inexpensive and accessible for quantification of changes in shape.
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Percepção da comunidade acadêmica da Universidade Federal de São Paulo - Campus Baixada Santista em relação ao uso indiscriminado e descarte inapropriado de fármacos e de produtos de cuidados pessoais
(Universidade Federal de São Paulo (UNIFESP), 2019-03-18) Noronha, Deborah Mendonça [UNIFESP]; Silva, Regina Cláudia Barbosa da [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Pharmaceuticals and personal care products (PPCPs) are the two major classes of emerging contaminants from urban sources, because they are produced and consumed in large quantities and they are introduced into the environment on large scale. They contribute to the contamination of water bodies, mainly due to their extensive use and inappropriate disposal, since in general the wastewater treatment plants are inefficient. Although, found in low concentrations, they can cause risks to human health and ecosystems. The aim of the present study was to evaluate, through a questionnaire, the perception of the academic community of Campus Baixada Santista of the Universidade Federal de São Paulo - Unifesp, regarding the indiscriminate use and inappropriate disposal of drugs and personal care products, as well as the knowledge about the damages caused by these practices, according to socioeconomic factors: occupational category, education level, age group and family income. The sample consisted of 847 individuals. The result indicated that 65.9% discard drugs inappropriately and that socioeconomic factors are related to this behavior. Proper disposal is more accomplished by individuals from Instituto do Mar (Imar) compared to individuals from the Instituto de Saúde e Sociedade (ISS), showing the lack of transversality of this theme in Campus Baixada Santista. Individuals who have lower schooling and age range are the ones who discard most inappropriately. There was no association with income. Concerning the perception of the participants regarding the damages that the indiscriminate use and the inappropriate disposal of medicines can cause, there was a statistically significant difference only in relation to the occupational category, pointing out more knowledge of the undergraduate students of the Imar in comparison to the following categories: lato sensu postgraduate students, ISS undergraduates students and ISS professors. We infer that the incorrect disposal of drugs is occurring due to the lack of knowledge about appropriate disposal, guidelines on how to dispose it properly and due to scarcity of the public campaigns clarifying the population, since the participants demonstrated knowledge about the damages caused by this behavior. Regarding the perception of environmental damage generated by the indiscriminate use and inappropriate disposal of personal care products, our results showed that individuals belonging to the Imar presented a higher perception in comparison to the ISS individuals, just as occurred in relation to the medications. Additionally, it was verified that individuals with lower age range and lower schooling show greater perception on this topic. There was no association with income. In conclusion, the identification of socioeconomic factors can contribute to the targeting of more efficient public campaigns in order to promote behavioral changes helping to reduce contamination by FPCPs in ecosystems. It is also necessary joint prevention activities between different sectors involved and more acting of the public power.
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Biocompatibilidade e potencial osteogênico de scaffolds manufaturados a partir de esponjas marinhas
(Universidade Federal de São Paulo (UNIFESP), 2019-03-29) Santos, Cintia Pereira De Goes [UNIFESP]; Granito, Renata Neves [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The marine sponges present interesting features, such as their three-dimensional architecture with interconnected pores, as well as their organic and inorganic components, which make them relevant candidates as biomaterials for the engineering of bone tissue. Therefore, this study aims to characterize and compare the in vivo osteogenic potential of scaffolds manufactured from two species of marine sponges, Dragmacidon reticulatum and Amphimedon viridis, both of Class Demospongiae. Initially, the marine sponges were collected, cut, lyophilized and sterilized to make the scaffolds. The scaffolds obtained were characterized by MEV, FTIR, XRD, EDS, besides the pH and mass degradation assay. Following, in vivo tests were performed using a scaffold implantation model in bone defect surgically created in rat tibiae. The animals were randomly divided into three groups: control group (GC), in which the bone defects were not filled with biomaterial, and the Dragmacidon reticulatum (GD) and Amphimedon viridis (GA) groups, in which the bone defects were filled with the scaffolds manufactured from the species Dragmacidon reticulatum and Amphimedon viridis, respectively. The animals were euthanized 15 days after surgery, the right tibias were used for biomechanical testing and the left tibia included in methyl methacrylate for histological and histomorphometric analysis of the non-decalcified bone tissue. In the characterization analyzes of the scaffolds, the SEM / EDS showed in both samples the presence of silica spurs, pores and the same chemical composition, the main elements being Si (silicon), O (oxygen) and C (carbon). There was a higher pore presence in a qualitative analysis, and a consequent greater degradation in the scaffolds of GD. pH analysis also revealed a greater variation in GD, with a more significant fall at the beginning of incubation (first three days). The XRD demonstrated the amorphous nature of scaffolds, with low crystallinity, for both species. The FTIR compared the functional groups and indicated a greater loss of organic matter in GD. In the histological analysis, the GC presented extensive formation of bone tissue at the edges of the lesion, with presence of osteoid tissue around it. The GD, when compared to GA, presented some points of neoformed bone tissue and a greater amount of osteoid tissue within the bone defect, always around the silica spicules. In turn, GA presented a fibrous capsule around the lesion, no formation of bone tissue, and a small amount of osteoid tissue at isolated sites within the defect. At the same time, in the histomorphometric analysis, GD presented a significantly higher percentage of osteoid tissue (OV / TV,%), as well as a significantly larger osteoblastic surface with respect to the bone surface (Ob.S / BS,%). In the biomechanical evaluation, there was no statistical difference between the studied species. Thus, the results suggest that the samples of the Dragmacidon reticulatum species, when compared to those of the species Amphimedon viridis, are potentially more suitable for the favoring of bone regeneration and, therefore, for use in tissue engineering.
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Avaliação da atividade de cisteinoproteases em brânquias de ostras do gênero Crassostrea sp. expostas a Clorotalonil
(Universidade Federal de São Paulo (UNIFESP), 2019-04-30) Utsumi, Giovanna Sayuri Azarias [UNIFESP]; Choueri, Rodrigo Brasil [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
After banning the use of organotin compounds as an active ingredient in antifouling paints, Chlorothalonil, a widely used fungicide in the agricultural sector, started to be commercialized worldwide as part of antifouling systems for use in ship hulls. Due to physicochemical characteristics in marine and estuarine environments, this biocide tends to associate with the sediment and may cause adverse effects on non-target organisms. Oysters are filtering sessile organisms that can be found in direct contact with the sediment and, therefore, have been widely used as bioindicators to monitor the environmental health of aquatic ecosystems. Currently, studies have resorted to established biomarkers to assess biological effects of Chlorothalonil, rather than developing specific biomarkers based on the mode of action of this biocide. In this context, the main objective of this study was to compare the activity of cysteinoproteases with biomarkers consecrated through exposure of oysters Crassostrea sp. to sediments fortified with Chlorothalonil, considering two exposure periods. Initially, responses of cathepsin B and cathepsin L proteases were similar to responses of some established biomarkers, such as lipid peroxidation and reduced glutathione, with a U-shaped dose-response. In contrast, glutathione peroxidase and DNA damage increased compared to acetone control, while acetylcholinesterase and glutathione S-transferase showed no significant difference. After 96h of exposure, a majority of the biological responses showed a tendency to reestablish cellular homeostasis in gills, thus suggesting that after the cathepsins action in the gills, the antioxidant defenses were inhibited, as a possible result of cellular homeostasis reestablishment. Taken together, the results of this study demonstrated that cathepsins B and L play important role in Cholothalonil detoxification and reestablishment of cellular homeostasis and, therefore, show a potential to be used as a biomarker for this biocide.
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Identificação molecular de candidatos a biomarcadores/bioindicadores de impactos antrópicos através de micro-organismos aquáticos em região estuarina da Baixada Santista
(Universidade Federal de São Paulo (UNIFESP), 2019-05-10) Garcia, Heitor [UNIFESP]; Gregoracci, Gustavo Bueno [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
The estuarine region of Santos and Sao Vicente is amongst the most impacted areas of Brazil. In this context, it is possible to include ahigh number of ships bound to the greatest port in Latin America, an industrial district located in Cubatão near the estuary, and lack of sanitation. Despite its great importance, surface water quality is impacted by inefficient sewage collection and treatment and by industrial effluents released in the river path. To evaluate the antropic impacts we employed a culture independent methodology (shotgun metagenomics) to seek potential biomarker and/or bioindicator microorganisms which could determine environmental contamination of the water column. The methodology employed here optimized the analysed information by using incremented good quality data, which was added to the files and annotated in MG-Rast. We analysed physical-chemical parameters along the relative picoplankton abundances to correlate organisms and environment. Santos presented the smaller species richness and biodiversity, when confronted with Sao Vicente and control site Cananeia, probably due to larger income of chemical contamination, which likely was limiting factor to many micro-organisms. All sampling sites in this work have shown impacts related to sewage release in the marine environment or lack of treatment, including control site of Cananeia, which did not differ significatively from Sao Vicente, near palafitas. The current beach water balneability index seems ineffective, given the presence of many pathogenic microorganisms which impact human health and are not detected by this approach. Co-occurrence correlation networks are compromised for several physical-chemical parameters, and several pathogenic species are present. This problematic may be connected to the presence of metals and organic contaminants, and the proliferation of key microorganisms identified here may be necessary to make bioremediation possible and to lessen environmental impact. Marinomonas sp. MWYL1 and Marinobacter hydrocarbonoclasticus species could be sentinel organisms for HPA contamination and maybe biorremediation. Enterococcus faecalis and Staphylococcus aureus species seem to tolerate elevated NO2levels. Thalassiossira pseudonana maybe an import species to represent environmental water quality. Species from the Pseudomonas genus may be used as biomarkers of nitrogen contamination or bioremediation, being able to transform those compounds. This work makes it possible to observe the relation between microbial species and several physical-chemical parameters and identify targets which could enable healthier and more complete correlation networks, and maybe provide tools for future bioremediation of this environment.
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Investigação do potencial protetor da alga gracilaria birdiae sobre ratos machos juvenis expostos a agrotóxicos em mistura
(Universidade Federal de São Paulo (UNIFESP), 2019-05-13) Cavalcante, Deborah Navit De Carvalho [UNIFESP]; Perobelli, Juliana Elaine [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Marine algae have been considered as nutritious food supplements with antioxidant potential. The red algae Gracilaria birdiae is considered one of the most promising of the several species, however, it’s still little studied in experimental models in vivo and focus has been given to the in vitro antioxidant activity of its fraction of sulphated polysaccharides. The use of antioxidant supplements may also be an important mitigating agent for the adverse effects of environmental pollutants on health, since many of them, such as agrochemical residues, cause oxidative damage to cellular components. Under real conditions, exposure does not occur to only one class of agrochemicals, but to complex mixtures of them, often potentializing their effects. In this context, the present study aims to characterize the macronutrients of the whole powder of the G. birdiae seawwed, as well as its carotenoid and flavonoid fractions, and investigate its protective potential in vivo in rats exposed to a mixture with the three agrochemicals found in food in Brazil: carbendazim, acephate and dithiocarbamate. For this, juvenile male rats were used as experimental model, from postnatal day 23 to 53, distributed in 4 groups: CTL (received, daily, only corn oil vehicle and Nuvilab CR1 ration plus water; MIS (received, daily, the mixture of the pesticides diluted in corn oil); GB (received, daily, the vehicle (corn oil), orally, by gavage, and the commercial G. birdiae red seaweed added to the 5% commercial feed Nuvilab CR1) and the GBMIS group (They received daily the mixture of pesticides, diluted in corn oil, orally, by gavage and the red seaweed G. birdiae added to the commercial feed Nuvilab CR1 at 5%). In the present study, powder bromatology G. birdiae showed a centesimal composition consistent with that previously reported in the literature, which includes high concentrations of the micronutrient Iron. On the other hand, the analysis of carotenoids and flavonoids indicated a low amount of these active compounds. In vivo study showed that the mixture of agrochemicals used, as well as the supplementation with G. birdiae, decreased the efficiency of the spermatogenic process in the testis, consequently impacting the epididymal morphology. The presence of oxidative DNA damage and the increase in pro-apoptotic tissue markers indicate the possible mechanism of reproductive and genetic toxicity exerted by both agrochemicals and supplementation with algae commercial powder. Thus, it is necessary to improve control over the processing standards of algae destined for food, which will certainly reflect in greater safety for human consumption, since the supplementation with the integral seaweed powder (only form available in the market ) appears to exert adverse health effects.
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Caracterização dos efeitos adversos de agroquímicos isolados ou combinados e estudo do potencial protetivo de suplemento polivitaminico em ratos machos juvenis
(Universidade Federal de São Paulo (UNIFESP), 2019-06-17) Garcia, Mariana Simoes [UNIFESP]; Perobelli, Juliana Elaine [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Agrochemicals are the most widely used synthetic chemicals in the world. They have the function of minimizing pests that interfere with the production of the vast majority of agricultural crops. Since 2009, Brazil is the country that uses the biggest amount agrochemicals in the world. Residues from these products contaminates food, soil and water, leading to environmental exposure for the entire world population. Under real conditions, this exposure occurs to complex mixtures of agrochemicals, which may establish synergistic or additive effects to each other, enhancing or adding the consequent health damage. An important pathway of these chemicals is oxidative damage to cellular components generated by excess of free radicals due to an imbalance between the production of these reactive molecules and the natural antioxidant system. In this way, the consumption of multivitamins supplements can be used as a way to increase the antioxidant defense of the organism, mitigating the adverse effects of these toxic agents. In fact, some studies have been investigated thevinjuries caused by chemical mixtures in experimental models, as well as the protective effect of antioxidant agents, but the vast majority uses adult individuals. In this context, the importance of juvenile / pre-pubertal toxicity studies has been highlighted in the recent literature because it is a period of great sensitivity during postnatal development. Given the scarcity of studies and the relevance of the issue, the present study aims to investigate whether pre-pubertal exposures at low doses of Carbendazim, Acefato and Mancozebe, because they are present in greater quantity in the diet of Brazilians, alone or in a mixture, cause immediate repercussions on the reproductive and endocrine parameters of juvenile male Wistar rats. In addition, it evaluates the potential protective effect of co-administration of multivitamins and minerals supplement. For this purpose, rats were exposed to the agrochemicals alone or in mixture (binary or triple) with or without supplementation, orally by gavage, from the postnatal day 23 to 53, when they were euthanized and evaluated for the histomorphometric parameters of testis and epididymis. Serum levels of steroid hormones were evaluated. In summary, the results indicate likely endocrine disrupting action of agrochemicals. Interestingly, isolated agrochemicals appear to exert more pronounced adverse effects than when administered in mixture. However, the group treated with multivitamin supplement did not present a significant protective effect as a form of protection against toxicological effects.
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Investigação das moléculas envolvidas na permeabilização das membranas e na regulação do processo de egresso de plasmodium falciparum na hemácia infectada
(Universidade Federal de São Paulo (UNIFESP), 2019-07-31) Melo, Hamille Rocha [UNIFESP]; Azevedo, Mauro Ferreira de [UNIFESP]; http://lattes.cnpq.br/1657599115711431; http://lattes.cnpq.br/6091182573007243; Universidade Federal de São Paulo (UNIFESP)
Introduction: In spite of the recent advances towards its control, malaria is still a burden to society, killing mainly children under five years of age. While successful treatment with total cure is available, drug resistance threatens patients care in the future and therefore, new antimalarial medicines are required. A potential target for chemotherapeutic intervention is host cell egress, a tightly regulated process that happens after parasites grow and multiply inside the human erythrocytes. Parasite enzymes are known to act in concert dismantling parasitophorous vacuole (PV) and red blood cell (RBC) membranes, but the hierarchy of their action is not completely understood. Objectives: The aims of this study were to establish a method that can be applied to identify compounds that affect egress and to investigate the sequence of the biochemical events involved. Methods: In order to synchronize parasites in two key moments that precede egress, P. falciparum 3D7 parasites were engineered to express endogenous calcium dependent protein kinase 5 (CDPK5) or protein kinase G (PKG) in fusion with a destabilization domain (DD). Expression of each DD tagged kinase is dependent on the ligand Shld-1 and its removal reversibly block egress. Expression of the sensitive reporter nanoluc (NLuc) either secreted do the PV or exported to the RBC was applied to quantify egress. Results: As expected, removal of Shld-1 nearly completely blocked egress in both transgenic lines and when Shld-1 was reintroduced to cultures previously synchronized with sorbitol and heparin at 4548 hours post invasion, Nluc activity in the supernatant increased in CDPK5-DD and PKG-DD cultures after 1 and 2 hours respectively. Microscopic and FACS analysis confirmed the increase in reporter activity correlated to egress. About a couple dozen compounds were evaluated. Egress of CDPK5-DD and PKG-DD were affected differently by a few compounds, demonstrating the method can be applied identify parasite enzymes and/or signalling pathways that act after PKG and before CDPK5. Conclusion: Remarkably, data provided by this study suggest a calpain and a few kinases such as PKA act temporally between PKG and CDPK5. Application of this protocol might allow the identification of many more compounds that affect egress and elucidating when their targets are required respectively to PKG and CDPK5 activation.
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Efeito do scaffold de biosilicato®/espongina no reparo de defeitos em tíbias de ratos
(Universidade Federal de São Paulo (UNIFESP), 2019-08-15) Fermino, Bianca Priscilla Dorileo [UNIFESP]; Renno, Ana Claudia Muniz [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
Bone repair is a highly complex process that includes the interaction of a series of biological events to restore tissue integrity. Nevertheless, some situations may result in delayed consolidation and even nonunion (SENA et al., 2005; MARSELL; EINHORN, 2010). This scenario has required the development of new therapeutic practices, such as the manufacture of composites that unite the properties of two or more materials, mainly due to the possibility they offer to mimic bone tissue (SIQUEIRA; ZANOTTO, 2011). Thus, the combination of Biosilicate® (BS) (which would represent the inorganic part of bone tissue) with an organic component, such as spongin (ESP) from marine sponges, could represent a therapy with increased osteogenic potential. Given the above, the objective of the present study was to evaluate the effect of the Biosilicate® Spongine (BS/ESP) scaffold on the repair process of induced bone defects in rat tibias. In this study, 30 rats allocated to the following experimental groups were used: CG (control); BS; and BS/ESP. The animals were submitted to the surgical procedure to perform the tibial bone defect and received the respective materials. After 15 days, the animals were euthanized and the samples collected for histopathological, morphometric – where three variables were investigated: ratio of neoformed bone tissue volume (BV) to defect volume (TV), (BV/TV), %), values for the number of osteoblasts per tissue area (N.Ob/T.Ar, mm2) and representation for the osteoblast surface as a percentage of bone surface (Ob.S/BS, %) – and immunohistochemistry analysis, which was performed qualitatively and semiquantitatively for RankL (activator of nuclear factor kappa-B ligand) and Runx2 (runt-related transcription factor-2) factors. Histopathological analysis indicated the presence of granulation tissue and few inflammatory cells at the bone defect site for all groups. Furthermore, there was a prevalence of bone trabeculae in the periphery of the defect for the biomaterials groups and signs suggestive of BS and BS/ESP degradation in the center of the bone defect. Regarding the morphometric analysis, no statistically significant difference was observed in any of the analyzed variables. Regarding qualitative immunohistochemical analysis, RankL immunostaining for CG was predominant in granulation tissue, osteocytes and bone marrow. For BS and BS/ESP, this immunostaining was more evident in granulation tissue and newly formed bone. Immunostaining for Runx2, on the other hand, was observed in all experimental groups in granulation tissue and newly formed bone. And for quantitative immunohistochemical analysis no statistically significant differences were observed between the studied groups for RankL and Runx2. It was concluded that the BS and BS/ESP scaffolds allowed the growth of bone trabeculae mainly at the edges of the defect. In addition, both materials showed a positive immunostaining for RankL and Runx2, mainly in granulation tissue and newly formed bone.
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Avaliação dos efeitos biológicos da acidificação dos oceanos associados a presença do fármaco losartan. Math
(Universidade Federal de São Paulo (UNIFESP), 2019-08-27) Guimaraes, Matheus Mascarin [UNIFESP]; Cesar, Augusto [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
In Brazil, deep oil exploration activities include the injection of CO2 to increase the pressure to remove oil from the wells, thus increasing the efficiency of the extraction system. This technique is used, among other reasons, to decrease CO2 concentrations in the atmosphere. However, accidental leakage of CO2 can result in several effects on the marine ecosystem, especially those related to the bioavailability of emerging pollutants. Since the last decade, several studies have reported the presence and effects of drugs in the marine environment, especially the antihypertensive class, such as losartan, which is mostly used by the elderly population. In addition, current estimates are for an increase of up to 100% of the elderly population over the next 30 years. However, there is little knowledge about the occurrence and physiological effects of losartan in marine invertebrates, especially regarding its behavior in the face of a possible ocean acidification scenario. In this context, the objective of this study was to evaluate the potential environmental impacts associated with the acidification of marine environments from the evaluation of bioavailability and toxicity at different levels of the biological organization of the compound losartan on marine organisms in water and sediment. Water and sediment samples were collected at 5 points around the Santos Submarine Sewage outfall (SSO) and 2 points around the Guarujá Submarine Sewage Outfall (GSO). Losartan was observed at all sampling points, with concentrations ranging from 1.37 - 7.63 ng/L in water and from 0.08 to 3.10 ng/g in sediments. In the results of the water toxicity test with Echinometra lucunter, both pH 8.0 and 7.6, the LOEC values were 100mg/L, whereas for pH 7.3 the value was 50mg/L, with EpH50 set forth at 7.35. In the sediment, a CEO of 10μg/g was obtained for pH 8.0, with effect from 1μg/g at pH 7.6 and 7.3, with EpH50 of 7.30. Regarding the trials with mussels Perna perna and Mytella guyanensis, effects were observed at environmentally relevant concentrations, with LOEC of 3000 ng/L and 3 ng/g, respectively. In addition, it was possible to observe the potentiation of the effects of losartan according to the decrease in pH. This study demonstrated that acidification of the oceans by itself causes effects on marine invertebrates, as well as influencing the toxicity of emerging pollutants. In this sense, there is a need to deepen the studies on drug-related toxicology and changes in pH in the marine environment.
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Efeitos neurobiológicos do tratamento com sakuranetina em camundongos.
(Universidade Federal de São Paulo (UNIFESP), 2019-09-27) Silva, Wilson Vicente Da [UNIFESP]; Ribeiro, Alessandra Mussi [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
It is recognized that extracts and substances obtained from natural products, such as plants and animal venom, can contribute to the development of model drugs. The use of medicinal plants for the treatment of various is the oldest and most traditional form of therapeutic model in different civilizations. Brazil has a vast plant biodiversity, resulting in a wide heterogeneity of bioactive chemicals that can be isolated and used for medicinal purposes. Many molecules isolated from plant extracts can have pharmacological effects on different biological tissues, such as antioxidant and neuroprotective action on nervous tissue. In this context, sakuranetin (SAK) is a plant-isolated flavonoid, and previous studies have shown that this compound has antiparasitic, anti-inflammatory and antioxidant activity. Thus, the aim of the present study was to investigate the potential anxiolytic and anticonvulsant effects of acute administration of SAK in mice. For this study male Swiss mice (3 months) were used. For the analysis of the effects of SAK administration, the animals received intracerebroventricular (i.c.v.) administration of SAK at 1, 10 or 20 mg/kg (SAK1, SAK10 or SAK20, respectively) doses and the behavior was evaluated in the open field test (CA, 20 min), and immediately after the animals were submitted to the elevated plus-maze test (LCE, 5 min). Anticonvulsant effect was evaluated, thus SAK (1 or 10 mg / kg, i.c.v.) was previously administered in mice and after 30 min was injected with bicuculline (BIC, 1 mg/kg). Our main results showed that animals treated with SAK1 and SAK10 traveled a greater distance and higher speed (p < 0.05). In addition, SAK10-treated animals increased the entries frequency in enclosed arms compared with control animals (p < 0.05). SAK10-treated animals increased latency for first seizure (p < 0.05), survival percentage (p < 0.05) and percentage of time between time to first seizure and death (p < 0.05). Taken together, these results suggest that acute treatment with low dose SAK promotes hyperlocomotion (1 mg/kg), and high dose (20 mg/kg) induces anxiogenic effect in animals. Furthermore, intermediate dose (10 mg/kg) induces anxiety-like and hyperlocomotor behavior in animals, but there is anticonvulsant effect. Our next steps involve performing immunohistochemistry analysis to understand what brain areas are active during these behaviors and after investigate action mechanism involved in anticonvulsant effect.

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