Validação de uma PCR em tempo real para o diagnóstico da doença de Aujeszky

Exportado OPUSMade available in DSpace on 2019-08-13T07:13:06Z (GMT). No. of bitstreams: 1 disserta__o_carolina_nonaka.pdf: 791721 bytes, checksum: 63623438a084a09c5e29f1a5ddbfce22 (MD5) Previous issue date: 5A doença de aujeszky (DA) é uma doença infecto-contagiosa de etiologia viral que se constitui em um importante obstáculo à exploração e ao comércio internacional de suínos. O SuHV-1 pertence a subfamília Alphaherpesvirinae e possui dentre outras características a capacidade de estabelecer infecções latentes nos neurônios sensoriais. Considerada como doença de notificação obrigatória pela OIE, está presente na lista de doenças de importância econômica e epidemiológica. Os sistemas de produção se tornaram mais intensivos e confinados, e consequentemente os riscos de disseminação de doenças também aumentaram. A adoção de estratégias e programas oficiais de controle e erradicação da DA, tendo em vista prevenir a disseminação da enfermidade, se faz necessário para evitar prejuízos extensos à exploração de suínos no Brasil. O presente trabalho teve como objetivo validar uma PCR em tempo real para o diagnóstico da DA e verificar o desempenho dos diferentes sistemas: SybrGreen I, Sonda de hibridização e Plexor. Para a verificação de desempenhos os seguintes critérios foram usados: Especificidade e Seletividade, Sensibilidade, Limite de detecção, Repetitividade e Reprodutibilidade e Robustez. O melhor desempenho foi alcançado utilizando os iniciadores e sonda desenhados para o alvo gB, este fato influenciou na tomada de decisão na escolha da metodologia a ser validada e disponibilizada para o Laboratório de Biologia Molecular do LANAGRO-MG, como ferramenta para o diagnóstico da DA no Brasil.Aujeszky's disease (AD) is an contagious infectious disease of viral etiology that constitutes a major obstacle to the exploration and international trade of swine. The Suid Herpesvirus 1 (SuHV-1) belongs to the subfamily Alphaherpesvirinae and among other features has the ability to establish latent infections in sensory neurons. Considered as a notifiable disease by the OIE, is present in the list of diseases of importance economic and epidemiology in swine. The production systems become more intensive and confined, and consequently the risk of spreading disease also increased. The adoption of strategies and public programs for control and eradication of AD, in order to prevent the spread of the disease, it is necessary to prevent extensive damage to the pig farm in Brazil. The aim of this work was to validate a real time PCR for the diagnosis of AD and to verify the performance of different systems: SybrGreen I, hybridization probe and Plexor. To check the performance of the following criteria were used: specificity and selectivity, sensitivity, limit of detection, repeatability and reproducibility and robustness. The best performance was achieved using the primers and probes designed to target gB, this fact influenced the decision-making in the choice of methodology to be validated and available in the Laboratory of Molecular Biology of the LANAGRO-MG, as a tool for the AD diagnostic in Brazil

Advances in Hepatic Tissue Bioengineering with Decellularized Liver Bioscaffold

The burden of liver diseases continues to grow worldwide, and liver transplantation is the only option for patients with end-stage liver disease. This procedure is limited by critical issues, including the low availability of donor organs; thus, novel therapeutic strategies are greatly needed. Recently, bioengineering approaches using decellularized liver scaffolds have been proposed as a novel strategy to overcome these challenges. The aim of this systematic literature review was to identify the major advances in the field of bioengineering using decellularized liver scaffolds and to identify obstacles and challenges for clinical application. The main findings of the articles and each contribution for technique optimization were highlighted, including the protocols of perfusion and decellularization, duration, demonstration of quality control—scaffold acellularity, matrix composition, and preservation of growth factors—and tissue functionality after recellularization. In previous years, many advances have been made as this technique has evolved from studies in animal models to human livers. As the field develops and this promising technique has become much more feasible, many challenges remain, including the selection of appropriate cell types for recellularization, route of cell administration, cell-seeding protocol, and scalability that must be standardized prior to clinical application

Exercise Training-Induced Changes in MicroRNAs: Beneficial Regulatory Effects in Hypertension, Type 2 Diabetes, and Obesity

MicroRNAs are small non-coding RNAs that regulate gene expression post-transcriptionally. They are involved in the regulation of physiological processes, such as adaptation to physical exercise, and also in disease settings, such as systemic arterial hypertension (SAH), type 2 diabetes mellitus (T2D), and obesity. In SAH, microRNAs play a significant role in the regulation of key signaling pathways that lead to the hyperactivation of the renin-angiotensin-aldosterone system, endothelial dysfunction, inflammation, proliferation, and phenotypic change in smooth muscle cells, and the hyperactivation of the sympathetic nervous system. MicroRNAs are also involved in the regulation of insulin signaling and blood glucose levels in T2D, and participate in lipid metabolism, adipogenesis, and adipocyte differentiation in obesity, with specific microRNA signatures involved in the pathogenesis of each disease. Many studies report the benefits promoted by exercise training in cardiovascular diseases by reducing blood pressure, glucose levels, and improving insulin signaling and lipid metabolism. The molecular mechanisms involved, however, remain poorly understood, especially regarding the participation of microRNAs in these processes. This review aimed to highlight microRNAs already known to be associated with SAH, T2D, and obesity, as well as their possible regulation by exercise training

Different methods of real-time PCR for detection of pseudorabies virus

ABSTRACT: Pseudorabies (PR) is a highly contagious viral disease of great animal health and economic importance in swine industry. The aim of this study was to evaluate different genomic regions, real-time PCR chemistries and equipment for the molecular diagnosis of PR. Eight primer pairs targeting four genes (gB, gC, gE, gD), three different qPCR chemistries (SybrGreen, hydrolysis probes and plexor) and two equipment (ABI7500, Rotorgene 3000) were evaluated. Oligonucleotides targeting gB using hydrolysis probes showed the best performance after evaluating efficiency (99%), the detection limit (10-1.5 TCID50 mL-1) and diagnostic sensitivity and; therefore, those primers were selected for performance verification factors such as repeatability, reproducibility and robustness (1.39% variance between days, 24% variance between analysts and 4.07% variance in analysis error). The qPCR standardized and validated in this research proved to be reliable for the diagnosis of PR and may be used in diagnostic laboratories that follow ISO 17025 and ISO 16140

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-08-14T12:25:36Z No. of bitstreams: 1 Evangelista AF Bone marrow-derived mesenchymal ....pdf: 3920676 bytes, checksum: c9784ec89d0678d1be3c53cf120dd471 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-08-14T12:46:12Z (GMT) No. of bitstreams: 1 Evangelista AF Bone marrow-derived mesenchymal ....pdf: 3920676 bytes, checksum: c9784ec89d0678d1be3c53cf120dd471 (MD5)Made available in DSpace on 2018-08-14T12:46:12Z (GMT). No. of bitstreams: 1 Evangelista AF Bone marrow-derived mesenchymal ....pdf: 3920676 bytes, checksum: c9784ec89d0678d1be3c53cf120dd471 (MD5) Previous issue date: 2018National Institutes of Health guide for the care and use of Laboratory animals (NIH, 8023) and the Institutional Animal Care and Use Committee FIOCRUZ (CPqGM 025/2011)Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilFederal University of Bahia. Pharmacy College. Salvador, BA, BrasilSão Rafael Hospital. Center of Biotechnology and Cell Therapy. Salvador, BA, BrazilFederal University of Recôncavo of Bahia. Feira de Santana, BA, BrazilSão Rafael Hospital. Center of Biotechnology and Cell Therapy. Salvador, BA, BrazilSão Rafael Hospital. Center of Biotechnology and Cell Therapy. Salvador, BA, BrazilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / São Rafael Hospital. Center of Biotechnology and Cell Therapy. Salvador, BA, BrazilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Federal University of Bahia. Pharmacy College. Salvador, BA, BrasilDiabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction. Methods: C57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses. Results: Diabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1β, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-β. Conclusions: The present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN

Generation of three control iPS cell lines for sickle cell disease studies by reprogramming erythroblasts from individuals without hemoglobinopathies

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2019-07-31T17:09:35Z No. of bitstreams: 1 Paredes, B.D. Generation of three... 2019.pdf: 1653990 bytes, checksum: 9764d1e4b334dd986d9f013cbe6b0931 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2019-07-31T17:21:24Z (GMT) No. of bitstreams: 1 Paredes, B.D. Generation of three... 2019.pdf: 1653990 bytes, checksum: 9764d1e4b334dd986d9f013cbe6b0931 (MD5)Made available in DSpace on 2019-07-31T17:21:24Z (GMT). No. of bitstreams: 1 Paredes, B.D. Generation of three... 2019.pdf: 1653990 bytes, checksum: 9764d1e4b334dd986d9f013cbe6b0931 (MD5) Previous issue date: 2019CNPq, CAPES, FAPESB, Ministry of Health, National Induced Pluripotent Stem Cell Biobank and the National Institute of Science and Technology for Regenerative Medicine.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, Brazil.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil.Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil.São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil / Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / São Rafael Hospital. D'Or Institute for Research and Education. Salvador, BA, Brazil.Sickle cell disease (SCD) is one of the most prevalent and severe monogenetic disorders. Previously, we generated iPS cell lines from SCD patients. Here, we generated iPS cell lines from three age-, ethnicity- and gender-matched healthy individuals as control cell lines. Cell reprogramming was performed using erythroblasts expanded from PBMC by a non-integrative method. SCD-iPSC controls expressed pluripotency markers, presented a normal karyotype, were able to differentiate into the three germ layers in embryoid body spontaneous differentiation and confirmed to be integration-free. The cell lines generated here may be used as matched healthy controls for SCD studies

Generation and characterization of transgenic mouse mesenchymal stem cell lines expressing hIGF-1 or hG-CSF

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2018-08-14T13:51:05Z No. of bitstreams: 1 Gonçalves GV Generation and characterization of transgenic mouse....pdf: 3184309 bytes, checksum: 06a39efddde966ed6d8c4f753691c630 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2018-08-14T14:05:27Z (GMT) No. of bitstreams: 1 Gonçalves GV Generation and characterization of transgenic mouse....pdf: 3184309 bytes, checksum: 06a39efddde966ed6d8c4f753691c630 (MD5)Made available in DSpace on 2018-08-14T14:05:27Z (GMT). No. of bitstreams: 1 Gonçalves GV Generation and characterization of transgenic mouse....pdf: 3184309 bytes, checksum: 06a39efddde966ed6d8c4f753691c630 (MD5) Previous issue date: 2018The National Council for Scientific and Technological Development (CNPq), The Foundation of Support for Research of the State of Bahia (FAPESB), and Funding Authority for Studies and Projects (FINEP).Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Salvador, BA, Brasil / National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, BrazilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, BrazilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Salvador, BA, Brasil / National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, BrazilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / National Institute of Science and Technology for Regenerative Medicine. Rio de Janeiro, RJ, BrazilMesenchymal stem cells (MSC) are promising tools in the fields of cell therapy and regenerative medicine. In addition to their differentiation potential, MSC have the ability to secrete bioactive molecules that stimulate tissue regeneration. Thus, the overexpression of cytokines and growth factors may enhance the therapeutic effects of MSC. Here we generated and characterized mouse bone marrow MSC lines overexpressing hG-CSF or hIGF-1. MSC lines overexpressing hG-CSF or hIGF-1 were generated through lentiviral vector mediated gene transfer. The expression of hG-CSF or hIGF-1 genes in the clones produced was quantified by qRT-PCR, and the proteins were detected in the cell supernatants by ELISA. The cell lines displayed cell surface markers and differentiation potential into adipocytes, osteocytes and chondrocytes similar to the control MSC cell lines, indicating the conservation of their phenotype even after genetic modification. IGF-1 and G-CSF transgenic cells maintained immunosuppressive activity. Finally, we performed a comparative gene expression analysis by qRT-PCR array in the cell lines expressing hIGF-1 and hG-CSF when compared to the control cells. Our results demonstrate that the cell lines generated may be useful tools for cell therapy and are suitable for testing in disease models

SARS-CoV-2 Detection via RT-PCR in Matched Saliva and Nasopharyngeal Samples Reveals High Concordance in Different Commercial Assays

Background: Self-collected saliva samples can increase the diagnostic efficiency and benefit healthcare workers, patient care, and infection control. This study evaluated the performance of self-collected saliva samples compared to nasopharyngeal swabs using three commercial kits for the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: Matched nasopharyngeal and saliva samples were collected from 103 patients with either asymptomatic or symptomatic COVID-19. Both samples were evaluated using three commercial kits (TaqCheck, Allplex, and TaqPath). To evaluate sample stability, viral RNA extraction was performed in the presence or absence of an RNA-stabilizing solution. Storage conditions, including the duration, temperature, and stability after freezing and thawing of the samples, were also evaluated. Results: All the saliva samples showed 100% concordance with the nasopharyngeal swab results using TaqCheck and Allplex kits, and 93% using TaqPath kit. No difference was observed in the samples that used the RNA-stabilizing solution compared to the group without the solution. The Ct values of the freeze–thawed samples after 30 days were higher than those on day 0; however, the results were consistent the fresh samples. Conclusion: The high concordance of SARS-CoV-2 detection via reverse transcription–polymerase chain reaction (RT-PCR) in matched saliva and nasopharyngeal samples using different commercial assays reinforces the concept that self-collected saliva samples are non-invasive, rapid, and reliable for diagnosing SARS-CoV-2 infection

Therapeutic effects of sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) in experimental chronic Chagas disease cardiomyopathy

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2017-08-16T16:44:20Z No. of bitstreams: 1 Vasconcelos JF Therapeutic effects....pdf: 5296745 bytes, checksum: 1f932cf358f4ef9b9c7ab2a388430048 (MD5)Approved for entry into archive by Ana Maria Fiscina Sampaio ([email protected]) on 2017-08-16T17:37:48Z (GMT) No. of bitstreams: 1 Vasconcelos JF Therapeutic effects....pdf: 5296745 bytes, checksum: 1f932cf358f4ef9b9c7ab2a388430048 (MD5)Made available in DSpace on 2017-08-16T17:37:49Z (GMT). No. of bitstreams: 1 Vasconcelos JF Therapeutic effects....pdf: 5296745 bytes, checksum: 1f932cf358f4ef9b9c7ab2a388430048 (MD5) Previous issue date: 2017National Council for Scientific and Technological Development (CNPq) and Bahia Research Foundation (FAPESB).Fundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Universidade Salvador. Escola de Ciências da Saúde. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, Brasil / Universidade Federal de Bahia. Instituto de Ciências da Saúde. Departamento de Bioquímica e Biofísica. Salvador, BA, BrasilUniversidade Federal do Rio de Janeiro. Instituto de Biofísica Carlos Chagas Filho. Centro de Ciências da Saúde. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, BrasilHospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilFundação Oswaldo Cruz. Instituto Gonçalo Moniz. Salvador, BA, Brasil / Hospital São Rafael. Centro de Biotecnologia e Terapia Celular. Salvador, BA, BrasilChagas disease cardiomyopathy is a parasite-driven inflammatory disease to which there are no effective treatments. Here we evaluated the therapeutic potential of N,N-dimethylsphingosine(DMS), which blocks the production of sphingosine-1-phosphate(S1P), a mediator of cellular events during inflammatory responses, in a model of chronic Chagas disease cardiomyopathy. DMS-treated, Trypanosoma cruzi-infected mice had a marked reduction of cardiac inflammation, fibrosis and galectin-3 expression when compared to controls. Serum concentrations of galectin-3, IFNγ and TNFα, as well as cardiac gene expression of inflammatory mediators were reduced after DMS treatment. The gene expression of M1 marker, iNOS, was decreased, while the M2 marker, arginase1, was increased. DMS-treated mice showed an improvement in exercise capacity. Moreover, DMS caused a reduction in parasite load in vivo. DMS inhibited the activation of lymphocytes, and reduced cytokines and NO production in activated macrophage cultures in vitro, while increasing IL-1β production. Analysis by qRT-PCR array showed that DMS treatment modulated inflammasome activation induced by T. cruzi on macrophages. Altogether, our results demonstrate that DMS, through anti-parasitic and immunomodulatory actions, can be beneficial in the treatment of chronic phase of T. cruzi infection and suggest that S1P-activated processes as possible therapeutic targets for the treatment of Chagas disease cardiomyopathy

Early detection of P.1 variant of SARS-CoV-2 in a cluster of cases in Salvador, Brazil

We report 3 cases of severe COVID-19 due to the SARS-CoV-2 P.1 lineage in a familial cluster detected in Salvador, Bahia–Brazil. All cases were linked to travel by family members from the state of Amazonas to Bahia in late December 2020. This report indicates the cryptic transmission of the SARS-CoV-2 P.1 lineage across Brazil and highlights the importance of genomic surveillance to track the emergence of new SARS-CoV-2 variants of concern