Produção e avaliação de modelos de cultivo celular para teste de RNAs guia para o sistema CRISPR-Cas9

Background: The CRISPR-Cas9 gene editing system has emerged as a powerful tool for targeted genetic modifications. This doctoral thesis aimed to optimize the CRISPR-Cas9 system for use in bovine mammary epithelial cells (MDBK) and embryos. Methods: Specific sgRNAs were designed and produced to target loci of interest for gene editing in MDBK cells. Additionally, stable Cas9- expressing MDBK cells were generated. In vitro evaluation of sgRNA activity was conducted, and mRNA reporters were utilized for transfection efficiency confirmation. Antibiotic selection was employed to promote stable Cas9 expression in human and bovine cell lines. The efficiency of gene editing in embryos was also evaluated through PCR and sequencing. Results: The in vitro evaluation of sgRNA activity in MDBK cells showed variations in editing efficiency, emphasizing the need for cell lines expressing Cas9 with relevant genomes. The mRNA reporter method demonstrated its usefulness for confirming transfection efficiency. Antibiotic selection successfully promoted stable Cas9 expression in human and bovine cell lines. Gene editing in embryos resulted in a total editing proportion of 48%, with the majority of mutant cells (35.5%) exhibiting a single-nucleotide deletion. Conclusion: The designed sgRNAs enabled gene editing in MDBK bovine cell lines, and the optimized CRISPR-Cas9 model was effective for editing the target loci in embryos. These findings contribute to the advancement of bovine genome engineering, offering potential applications in future studies involving the CRISPR-Cas9 system for bovine genome editing.Contexto: O sistema de edição gênica CRISPR-Cas9 tem se mostrado uma ferramenta poderosa para modificações genéticas direcionadas. Esta tese de doutorado buscou otimizar o sistema CRISPR-Cas9 para utilização em linhagens celulares bovinas e embriões. Métodos: Foram desenhados e produzidos sgRNAs específicos visando loci de interesse para edição gênica em células epiteliais mamárias bovinas (MDBK). Adicionalmente, células MDBK expressando estavelmente a proteína Cas9 foram geradas. A atividade dos sgRNAs foi avaliada in vitro, e RNA mensageiros (mRNA) reportadores foram utilizados para confirmar a eficiência da transfeção. A seleção por antibiótico foi empregada para promover a expressão estável da Cas9 em linhagens celulares humanas e bovinas. A eficiência da edição gênica em embriões também foi avaliada por meio de PCR e sequenciamento.Resultados: A avaliação in vitro da atividade dos sgRNAs em células MDBK demonstrou variações na eficiência de edição, destacando a necessidade de linhagens celulares expressando a Cas9 com genomas relevantes. O método do mRNA reportador demonstrou sua utilidade para confirmar a eficiência da transfeção. A seleção por antibiótico promoveu com sucesso a expressão estável da Cas9 em linhagens celulares humanas e bovinas. A edição gênica em embriões resultou em uma proporção total de edição de 48%, sendo a maioria das células mutantes (35,5%) apresentando uma deleção de um nucliotídeo. Conclusão: Os sgRNAs desenhados permitiram a edição gênica em linhagens celulares bovinas MDBK, e o modelo otimizado do sistema CRISPR-Cas9 mostrou-se eficaz para editar os loci alvo em embriões. Esses resultados contribuem para o avanço da engenharia do genoma bovino, oferecendo aplicações potenciais em futuros estudos envolvendo o sistema CRISPR-Cas9 para a edição do genoma bovino

Control of contaminants during introduction and establishment of Bambusa vulgaris in vitro

The aim of this work was to test techniques to reduce microbial contamination in the phases of introduction and establishment of the in vitro cultivation of Bambusa vulgaris through two experiments. The first experiment was carried out in a completely randomized design using a factorial arrangement (pre-treatment of nodal segments using or not a solution of Derosal 500 SC® and Chloramphenicol × culture medium with half or full concentration of salts × culture medium with presence or absence of sucrose × culture medium with presence or absence of Plant Preservative MixtureTM). In a second experiment, carried out in a completely randomized design, the effect of different fungicides associated to Chloramphenicol in a liquid culture medium was tested. It was possible to verify that the isolated effects of the pre-treatment by immersion of the nodal segments in a solution of 4 mL L-1 of Derosal 500 SC® and 200 mg L-1 of Chloramphenicol for 30 minutes and explants placed in a sucrose-free medium reduced fungal contamination. In the second experiment, the treatment that reduced fungal contamination corresponded to explants placed for seven days in a liquid medium with half the concentration of salts, sucrose-free, with 2 mL L-1 of Plant Preservative MixtureTM and with 4 mL L-1 of  Derosal 500 SC® and 200 mg L-1 of Chloramphenicol

Spatial and temporal variability of soil CO2 flux in sugarcane green harvest systems

The sugarcane green harvest system, characterized by mechanized harvesting and the absence of crop burning, affects soil quality by increasing crop residue on the soil surface after harvest; thus, it contributes to improving the physical, chemical, and microbiological properties and influences the soil carbon content and CO2 flux (FCO2). This study aimed to evaluate the spatial and temporal variability of soil FCO2 in sugarcane green harvest systems. The experiment was conducted in two areas of sugarcane in São Paulo, Brazil: the first had a 5-year history of sugarcane green harvest (SG-5) and the second had a longer history of 10 years (SG-10). The temporal FCO2 were evaluated in the dry and rainy periods, and spatial variability in the dry period, and related to soil chemical and physical properties, including organic C porosity, bulk density, soil penetration resistance, mean weight diameter of soil aggregates, clay, P, S, Ca, Mg and Fe. The temporal variability indicated no differences between the dry and rainy periods in SG-10, while in SG-5 soil moisture was increased by 33 % in the rainy period. The spatial variability indicated a different pattern from the temporal one, where FCO2 in SG-10 was correlated with soil temperature, air-filled pore space, total porosity, soil moisture, and the Ca and Mg contents; in the SG-5 area, FCO2 was correlated with soil mean weight diameter of soil aggregates and the sulfur content.4

As mulheres encarceradas e o dilema em exercer o papel de mãe

A assistência às mulheres encarceradas é um dever do Estado para prevenir o crime e facilitar sua integração na sociedade, garantindo os direitos humanos. Este estudo analisa a assistência à saúde fornecida às mulheres encarceradas e as dificuldades que enfrentam no ambiente prisional durante o período de gravidez e pós-parto. As discussões fazem parte de uma pesquisa que considera outros estudos sobre o tema. O gênero foi escolhido como um elemento central dessa pesquisa devido à sua relevância na história da criminalidade feminina. Pesquisas mostram que estudar a criminalidade praticada por mulheres é mais desafiador do que a dos homens, não apenas porque as mulheres cometem menos crimes, mas também pela escassez de casos, o que dificulta a pesquisa. Em relação à punição, observa-se uma estreita relação com o gênero, com penas direcionadas exclusivamente às mulheres. O artigo critica a situação das mães encarceradas, crianças e fetos em um ambiente que não favorece seu desenvolvimento integral. A pesquisa consiste em uma revisão bibliográfica, explorando fontes online, legislação brasileira e cartilhas sobre a situação das mães e filhos encarcerados

Odontogenic cysts : demographic profile in a Brazilian population over a 38-year period

Objective: To determine the distribution of odontogenic cysts diagnosed histologically over a period of 38 years in a Brazilian population according to age, gender and site affected and to compare these data with previously reported studies from other countries. Study design: A total of 1019 cases of odontogenic cysts diagnosed between 1970 and 2007 were studied. Clinical features obtained from the patient records and microscope slides were reviewed according to the 1992 World Health Organization classification. Results: The mean age was 31.0 years, and there was a predominance of females. The most frequent odontogenic cysts were radicular cysts (61.4%), followed by dentigerous cysts (20.1%) and odontogenic keratocysts (6.4%). Radicular cysts were more frequent in females (62.0%), and the maxillary teeth were the site most commonly involved (63.05%). The peak incidence of dentigerous cysts occurred in the second decade of life, with the posterior region of the mandible being the site most affected (46.3%), followed by the anterior region of the maxilla (27.8%). Odontogenic keratocysts showed a peak incidence between the third and fourth decades of life and predominance among females. The posterior region of the mandible was the site most frequently affected (65.6%). Conclusion: The present results showed a similar frequency of odontogenic cysts in this Brazilian population and other populations around the world, with inflammatory cysts being identified as the most frequent odontogenic cyst. Radicular cysts, dentigerous cysts, and odontogenic keratocysts are the most common cystic lesions, accounting for 87.9% of all odontogenic cysts

Marketing Approach of Brazilian Wind Energy Sector

Prospects for the wind energy market have proposed changes of focus to managerial issues. The objective of this article is to map the specific market factors from Brazilian wind energy industry, in order to develop reflections and considerations on the subject, towards to the managerial, strategic and commercial development of the sector. Through an exploratory methodology in empirical format, and by a SWOT analysis of Telescopic Observations Strategic Framework, were found results that show funding and grants determined by Brazilian government, as the big question of the wind energy industry marketing, allowing the economic viability of wind energy projects. Further, it appears that the wind energy industry is eager to investments and has great potential for new business, but there are problems within the producing companies which that have to be assessed, such as the competitiveness capability, the high equipment costs, the installation locations limitations and lack of specialized employees with specific skills and capacities

Competitiveness and Innovation in the New Products Development Process: A Case Study in a Brazilian Chemical Industry

Demands for higher production capacity and the increasing competition in various sectors of the Brazilian economy have required competitive levels more hard, which demanding tools, techniques and efficient processes. Within moderns business, the new products development process is highlighted as competitive differential in the organizational environment, in a global and intense format. In Brazil, although the researches are becoming frequent, studies on the new products development process has been newness conceptual in the economic sectors and industries. This shows the need for research that takes into consideration the factors considered impactful addressed by business institutions and practices used by them in the new products development process. Thus, this following work aims to present the new product development process in a Brazilian company that operates on the chemical industry, in which we aimed identify the responsible factors for the prominent business/financial success and of products developed by the company

Novel parvoviruses from wild and domestic animals in Brazil provide new insights into parvovirus distribution and diversity

Parvoviruses (family Parvoviridae) are small, single-stranded DNA viruses. Many parvoviral pathogens of medical, veterinary and ecological importance have been identified. In this study, we used high-throughput sequencing (HTS) to investigate the diversity of parvoviruses infecting wild and domestic animals in Brazil. We identified 21 parvovirus sequences (including twelve nearly complete genomes and nine partial genomes) in samples derived from rodents, bats, opossums, birds and cattle in Pernambuco, São Paulo, Paraná and Rio Grande do Sul states. These sequences were investigated using phylogenetic and distance-based approaches and were thereby classified into eight parvovirus species (six of which have not been described previously), representing six distinct genera in the subfamily Parvovirinae. Our findings extend the known biogeographic range of previously characterized parvovirus species and the known host range of three parvovirus genera (Dependovirus, Aveparvovirus and Tetraparvovirus). Moreover, our investigation provides a window into the ecological dynamics of parvovirus infections in vertebrates, revealing that many parvovirus genera contain well-defined sub-lineages that circulate widely throughout the world within particular taxonomic groups of hosts

Imunoterapia de CAR T-Cells Anti-CD19 no tratamento de leucemia linfoblástica aguda refratária/recidivante. Revisão sistemática e metanálise

Introdução: A Leucemia Linfoblástica Aguda (LLA) é a neoplasia maligna mais prevalente em crianças; entretanto, quando se torna refratária/recidivante (R/R) as possibilidades de cura são praticamente nulas. Objetivos: Analisar a eficácia da imunoterapia de Receptores de Antígenos Quiméricos anti-CD19 no tratamento da LLA R/R, fornecendo evidências sobre a efetividade e segurança da terapia para o grupo analisado. Métodos: O estudo consistiu em uma revisão sistemática e metanálise baseada em artigos indexados. As pesquisas foram realizadas com os termos: “acute lymphoblastic leukemia”, “CAR T”, and “CD19-specific chimeric antigen receptor”. Resultados: Dos 94 artigos obtidos, apenas 18 atenderam inicialmente aos critérios de inclusão e foram selecionados para revisão, totalizando 637 pacientes. Assim, observou-se nas respostas que aproximadamente 81% dos pacientes obtiveram resposta completa; 7% não responderam; a neoplasia recidivou em 17% dos casos; e 6,1% dos pacientes morreram. Os principais efeitos colaterais encontrados foram síndrome de liberação de citocinas, síndrome de liberação grave de citocinas e neurotoxicidade, presentes em 36,3%, 29% e 24% dos pacientes, respectivamente. Conclusão: A imunoterapia com células CAR T anti-CD19 é uma terapia eficaz, sendo capaz de produzir altas taxas de remissão completa no tratamento de LLA R / R.Introduction: Acute Lymphoblastic Leukemia (ALL) is the most prevalent malignancy in children; however, when the neoplasm becomes refractory/relapses (R/R) the cure possibilities are practically null. Objectives: To analyze the Anti-CD19 Chimeric Antigen Receptors (CAR) T-Cells immunotherapy efficacy in the treatment of R/R ALL, providing evidence about the efficacy and safety of the therapy for the analyzed group. Methods: The study consisted of a systematic review and meta-analysis based on the analysis of indexed articles. The searches were carried out with the terms: “acute lymphoblastic leukemia”, “CAR T”, and “CD19-specific chimeric antigen receptor”. Results: Only 18 of the 94 articles obtained initially met the inclusion criteria and were selected for review, totaling 637 patients. Thus, it was observed in the responses that approximately 81% of the patients achieved a Complete Response; 7% did not respond; the neoplasm relapsed in 17% of the cases; and 6.1% of the patients died. The main side effects found were Cytokine Release Syndrome (CRS), Severe Cytokine Release Syndrome, and Neurotoxicity, present in 36.3%, 29%, and 24% of patients, respectively. Conclusion: Anti-CD19 CAR T-Cells immunotherapy is an effective therapy, capable of producing high rates of complete remission in R/R ALL treatment