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- ItemAcesso aberto (Open Access)Avaliação da carga viral de SARS-CoV-2 após oligoterapia contra a região compreendida pela Transcriptional Regulatory Sequence (TRS)(Universidade Federal de São Paulo, 2022-11-10) Santos, Gabriela Vasconde dos [UNIFESP]; Janini, Luiz Mário Ramos [UNIFESP]; http://lattes.cnpq.br/5713863164263481; http://lattes.cnpq.br/7258861315725750A pandemia da COVID-19 tem sido um enorme desafio em saúde pública para o mundo todo. Ao longo dos últimos 2 anos, estima-se que 6,55 milhões de indivíduos tenham morrido devido à COVID-19, sobretudo nos EUA, Índia e Brasil. As manifestações clássicas dos casos graves da COVID-19 incluem sintomas aéreos superiores e angústia ao respirar. Internamente, é observado um quadro inflamatório e protrombótico intenso que leva à internação e dano tecidual pulmonar e renal que eventualmente culmina em óbito. As principais estratégias para evitar a doença consistem em medidas profiláticas não farmacológicas, com destaque para o uso de máscaras e distanciamento social, além de adoção a esquema vacinal. Uma vez contaminado, o paciente pode fazer uso de antinflamatórios corticoesteróides, imunobiológicos e medicamentos antivirais, todos com eficácia comprovada, porém, limitada. O SARS-CoV-2 é um coronavírus com semelhanças aos coronavírus responsáveis pela MERS e SARS – Suas Transcriptional Regulatory Sequences são altamente conservadas e representam um bom alvo terapêutico para atividade antiviral. Sendo assim, o objetivo deste trabalho foi desenhar e selecionar oligonucleotídeos capazes de alinhar com a TRS do SARS-CoV-2 e avaliar a atividade antiviral e citotoxicidade destes em células Vero E6. Os ensaios realizados demonstraram atividade antiviral de alguns oligonucleotídeos construídos, mesmo em concentrações baixas (a partir de 15.62 nM). O mesmo oligonucleotídeo, em um ensaio para citotoxicidade, não diminuiu a viabilidade celular da cultura de células Vero E6 em uma concentração de até 1000 nM. O oligonucleotídeo ainda diminuiu as taxas de cópias virais e a replicação do vírus. Embora ensaios complementares sejam necessários, os dados aqui apresentados indicam que a TRS do SARS-CoV-2 pode ser um alvo terapêutico valioso, além de ser de suma importância para a biologia desse vírus.
- ItemSomente MetadadadosCharacterization and role of the 3-methylglutaconyl coenzyme A hidratase in Trypanosoma brucei(Elsevier Science Bv, 2017) De Lima-Stein, Mariana Leão [UNIFESP]; Icimoto, Marcelo Yudi [UNIFESP]; Levatti, Erica Valadares de Castro [UNIFESP]; Oliveira, Vitor [UNIFESP]; Straus, Anita Hilda [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Trypanosoma brucei, the agent of African Trypanosomiasis, is a flagellated protozoan parasite that develops in tsetse flies and in the blood of various mammals. The parasite acquires nutrients such as sugars, lipids and amino acids from their hosts. Amino acids are used to generate energy and for protein and lipid synthesis. However, it is still unknown how T. brucei catabolizes most of the acquired amino acids. Here we explored the role of an enzyme of the leucine catabolism, the 3-methylglutaconyl-Coenzyme A hydratase (3-MGCoA-H). It catalyzes the hydration of 3-methylglutaconyl-Coenzyme A (3-MGCoA) into 3-hydroxymethylglutaryl-Coenzyme A (3-HMGCoA). We found that 3-MGCoA-H localizes in the mitochondria) matrix and is expressed in both insect and mammalian bloodstream forms of the parasite. The depletion of 3-MGCoA-H by RNA interference affected minimally the proliferation of both forms. However, an excess of leucine in the culture medium caused growth defects in cells depleted of 3-MGCoA-H, which could be reestablished by mevalonate, a precursor of isoprenoids and steroids. Indeed, procyclics depleted of the 3-MGCoA-H presented reduced levels of synthesized steroids relative to cholesterol that is scavenged by the parasite, and these levels were also reestablished by mevalonate. These results suggest that accumulation of leucine catabolites could affect the level of mevalonate and consequently inhibit the sterol biosynthesis, required for T. brucei growth. (C) 2017 Elsevier B.V. All rights reserved.
- ItemAcesso aberto (Open Access)A Interleucina 1-beta mostra uma ação protetora na fase aguda do modelo de epilepsia induzido pela pilocarpina(Liga Brasileira de Epilepsia (LBE), 2010-09-01) Pascoal, Vinícius d b; Marchesini, Rafael Breglio; Matos, Anabela hb; Conte, Fábio Frangiotti; Pereira, Tiago Campos; Gilioli, Rovilson; Malheiros, Jackeline Moraes [UNIFESP]; Polli, Roberson Saraiva [UNIFESP]; Buzzá, Hilde H; Tannús, Alberto; Covolan, Luciene [UNIFESP]; Cavalheiro, Esper Abrão [UNIFESP]; Velloso, Lício Augusto; Cendes, Fernando; Lopes-Cendes, Íscia; Universidade Estadual de Campinas (UNICAMP); Universidade de São Paulo (USP); Universidade Federal de São Paulo (UNIFESP)INTRODUCTION: There is contradictory information regarding the of effects il1β and il1rn in epilepsy. We aimed to evaluate the effect of silencing both genes in the acute phase of the pilocarpine-induced epilepsy model. METHODS: We used RNA interference in order to achieve gene silencing. RESULTS: We obtained significant gene silencing in the central nervous system. In addition, we observed phenotypic effects including differences in mortality rates of animals 5 days after pilocarpine injections. CONCLUSION: Our results indicate that il1β seems to have a protective effect in the acute phase of the pilocarpine-induced epilepsy model.
- ItemAcesso aberto (Open Access)Proposta de modelo in vitro de Doença Hepática Gordurosa Não-Alcoólica (DHGNA): Silenciamento do fator de crescimento TGF-a em células AML-12.(Universidade Federal de São Paulo, 2023-07-10) Tamashiro, Juliana Almeida [UNIFESP]; Christoffolete, Marcelo Augusto; http://lattes.cnpq.br/0613044471218500; http://lattes.cnpq.br/0127355353452898Introdução: A Doença Hepática Gordurosa Não Alcoolica é uma doença que acomete cerca de 25% da população mundial, caracterizada pelo acúmulo de triacilglicerídeos (TAG) no citoplasma dos hepatócitos, podendo evoluir a esteatohepatite, cirrose e hepatocarcinoma. Uma das vias que contribui para o acúmulo lipídico é a de novo lipogenesis (DNL), estimulada pelo alto consumo de frutose de alimentos ultraprocessados. A linhagem celular AML-12, de hepatócito de camundongo, é utilizado para o estudo da doença. No entanto, resultados prévios do nosso grupo, ainda não publicados, demonstram que ao invés da expressão esperada de Glicoquinase (GCK), a isoenzima expressa é a Hexoquinase II (HK2), impactando diretamente no metabolismo de glicose e consequentemente, na de novo lipogenesis. Materiais e Métodos: Foi construído através de clonagem, um vetor plasmidial contendo um short hairpin RNA (shRNA) direcionado ao RNA mensageiro (mRNA) do Transforming Growth Factor alpha (TGF-ɑ) a fim de realizar um silenciamento por interferência por RNA deste gene, responsável pela imortalização da linhagem AML-12. O estabelecimento do protocolo foi testado através do ensaio de beta-galactosidase. Resultados: A partir do seqüenciamento pelo método de Sanger, foi possível confirmar a obtenção correta do plasmídio contendo o inserto de shRNA para o gene do TGFα. Conclusão: É possível concluir que as modificações propostas no protocolo do fabricante foram decisivas para a obtenção do plasmídio de silenciamento.
- ItemAcesso aberto (Open Access)Proteínas quinases envolvidas na regulação do estresse em Trypanosoma(Universidade Federal de São Paulo (UNIFESP), 2010-03-31) Jesus, Teresa Cristina Leandro de [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Protozoa of the genus Trypanosoma have a complex life cycle alternating between vertebrate and invertebrate hosts. The adaptation to different environmental conditions requires rapid changes in gene expression to fill up the morphological and metabolic requirements for survival. Very little is known about the mechanisms that control these changes and the signaling pathways involved. As in these organisms the control of gene expression occurs at post-transcriptional level, in this work we decided to investigate the function of protein kinases involved in the control of protein synthesis and growth of these parasites. In several eukaryotes TOR (target of rapamycin) protein kinases are involved in protein synthesis control and cell growth in response of the availability of nutrients or growth factors. By searching T. brucei genomic database we found four candidates for TOR (TbTOR1, TbTOR2, TbTOR-like1 and TbTOR-like 2). Two TOR complexes were previously described in T. brucei (TbTORC1 and TbTORC2). In the first chapter of this thesis we study: TbTOR-like 1 and compared it with TbTOR2. TbTOR-like 1 is not present in any of the TORC complexes and has a PDZ domain not found in any of other TORs of T brucei, or other eukaryotes. It is located cytosolic granules that migrate to the cell periphery after hyperosmotic stress. Depletion TbTOR-like 1 causes a progressive inhibition of cell growth, generating enlarged cells that accumulate in S/G2 phase of the cell cycle. These cells also show increased number of acidocalcisomes and augmented levels of polyphosphate and pyrophosphate. These data indicate that TbTOR-like seems to be involved in controlling cell growth and biogenesis of acidocalcisomes responding to osmotic changes in the medium. In the second chapter of the thesis we studied protein kinases involved in protein synthesis control through the phosphorylation of the subunit of the eukaryotic translation initiation factor 2 (eIF2α).These kinases are activated by different types of stress. T. brucei encodes three potential eIF2α protein kinases (TbeIF2K1, K2 and K3). We studied more specifically the K2. We showed that it is a transmembrane glycoprotein located in the region of the flagellar pocket in both forms of T. brucei, and in the endosomal compartments of Trypanosoma cruzi. These endosomal compartments are known as reservosomes and they are formed only in the parasite’s stage that li ves in the digestive tract lumen of the insect vector. This fact suggests that in both parasites this protein kinase may be acting as a sensor in the transport of nutrients and proteins. In conclusion we revealed the existence of at least two mechanisms by which trypanosomes perceive and resist to environmental changes during their life cycle.
- ItemAcesso aberto (Open Access)Rmcystatin3, a cysteine protease inhibitor from Rhipicephalus microplus hemocytes involved in immune response(Elsevier B.V., 2014-11-01) Lu, Stephen [UNIFESP]; Soares, Tatiane Sanches [UNIFESP]; Vaz Junior, Itabajara S.; Lovato, Diogo Ventura [UNIFESP]; Tanaka, Aparecida Sadae [UNIFESP]; Universidade Federal de São Paulo (UNIFESP); Univ Fed Rio Grande do SulThe Rhipicephalus microplus tick is responsible for losses in the livestock production estimated in 2 billions USD. Despite its economical importance the knowledge in tick's physiology is sparse. in order to contribute to this scenario we describe the characterization of a cysteine proteinase inhibitor named Rmcystatin-3. Purified recombinant Rmcystatin-3 was able to inhibit cathepsin L (Ki = 2.5 nM), BmCl1 (Ki = 1.8 nM) and cathepsin B (Ki = 136 nM). Western blot and quantitative PCR analysis revealed the presence of Rmcystatin-3 in fat body, salivary gland but mainly in hemocytes. the mRNA levels of Rmcystatin-3 during bacterial challenge are drastically down-regulated. in order to define the Rmcystatin-3 possible role in tick immunity, the cystatin gene was knockdown by RNA interference with and without Escherichia coli infection. Our results showed that the Rmcystatin-3 silenced group was more immune competent to control bacterial infection than the group injected with non-related dsRNA. Taking together, our data strongly suggested an important role of Rmcystatin-3 in tick immunity. (C) 2014 Elsevier B.V. and Societe francaise de biochimie et biologie Moleculaire (SFBBM). All rights reserved.