Kaio César Simiano Tavares

Dengue and Zika, arboviruses are common in Brazil and present with similar clinical symptoms making specific diagnoses difficult. Therefore, a Duplex One-step RT-PCR method for dengue and zika virus was developed and validated in vitro and in silico, based on the dengue's NS1 region and zika's Envelope region.

The imprinted H19 long non-coding RNA, a knowing oncofetal gene, presents a controversial role during the carcinogenesis process since its tumor suppressor or oncogenic activity is not completely elucidated. Since H19 lncRNA is involved in many biological pathways related to tumorigenesis, we sought to develop a non-cancer lineage with CRISPR-Cas9-mediated H19 knockdown (H19-) and observe the changes in a cellular context. To edit the promoter region of H19, two RNA guides were designed, and the murine C2C12 myoblast cells were transfected. H19 deletion was determined by DNA sequencing and gene expression by qPCR. We observed a small deletion (~ 60 bp) in the promoter region that presented four predicted transcription binding sites. The deletion reduced H19 expression (30%) and resulted in increased proliferative activity, altered morphological patterns including cell size and intracellular granularity, without changes in viability. The increased proliferation rate in the H19- cell seems to facilitate chromosomal abnormalities. The H19- myoblast presented characteristics similar to cancer cells, therefore the H19 lncRNA may be an important gene during the initiation of the tumorigenic process. Due to CRISPR/Cas9 permanent edition, the C2C12 H19- knockdown cells allows functional studies of H19 roles in tumorigenesis, prognosis, metastases, as well as drug resistance and targeted therapy.

Com o presente trabalho, objetivou-se estudar a influência da somatotropina recombinante bovina (rbST) sobre a quantidade e qualidade de oócitos aspirados in vivo, produção embrionária e expressão gênica dos fatores de crescimento semelhantes à insulina (IGFs) tipos I e II em embriões produzidos in vitro de fêmeas bovinas da raça Nelore. Cinco vacas foram tratadas com duas administrações de 2mL de solução salina como placebo, com intervalo de catorze dias entre elas, sendo a primeira administração realizada dezenove e a segunda cinco dias antes das aspirações foliculares; decorridos trintas dias destas aspirações, em sistema crossover, o mesmo protocolo foi utilizado nesses cinco animais sendo, neste caso, a solução salina substituída por 500mg de rbST, com o intuito de efetuar-se comparações entre os tratamentos. A partir dos oócitos recuperados, realizou-se a produção in vitro de embriões (PIVE), sendo todas as etapas que envolvem este processo analisadas e com posterior realização de reações em cadeia da polimerase em tempo real (qPCRs) para expressões gênicas de IGF-I e IGF-II nos blastocistos produzidos. Observou-se que a rbST, ainda que numericamente tenha influenciado positivamente sobre os parâmetros estudados, não exerceu influência significativa sobre a quantidade e qualidade de oócitos aspirados in vivo, produção embrionária e expressão gênica de IGF-I e IGF-II em embriões produzidos in vitro de fêmeas bovinas da raça Nelore.

Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

Introduction: breast cancer (BC) is the most common tumor and the leading cause of cancer-related death among the female populationworldwide. Polymorphisms genetics of ABCB1 gene contributed to breast cancer susceptibility and interindividual differences inchemotherapy response. Objectives: to evaluate the association between the ABCB1 C3435T gene polymorphism (SNPs) with theresponse to neoadjuvant chemotherapy in women with breast cancer. Methodology: this study included 32 female patients whoreceived neoadjuvant chemotherapy. The polymorphisms were genotyped through real-time allele-specific polymerase chain reaction(PCR). The statistical analysis was performed using the Fisher’s exact test or Pearson’s chi-square test in the Statistical Package forSocial Sciences (SPSS) version 20.0 software. Results: the genotypes found for the C3435T polymorphism were in Hardy-Weinbergequilibrium and their genotypic distributions were CC= 10 (31.1%), CT= 14 (43.8%), and TT= 08 (25.0%) with χ2: 0.86 and p-value >0.05. Allele frequencies were C = 0.54 and T = 0.46. There were no significant statistical differences between genotypes considering theresponse to neoadjuvant chemotherapy and immunohistochemistry; the presence of the T allele was associated with worsen axillarystatus response to neoadjuvant chemotherapy. Conclusion: no definite association between the presence of C3435T polymorphismand the response to neoadjuvant chemotherapy was observed. Further studies in Brazil involving larger samples will contribute tovalidating the results of this study.

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells—GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A—0%, protocol B—93%, protocol C—13.33%, P TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

Several assays can be used to diagnose a patient with COVID-19. The polymerase chain reaction (PCR) technique is a molecular assay capable of detecting SARS-CoV-2 viral RNA with high specificity during the acute phase of infection. A wide variety of primers and probes were developed to detect this virus, mainly targeting the following genomic regions: ORF1ab, envelope genes (E), RNA-dependent RNA polymerase (RdRP), spike protein (S), and nucleocapsid (N)⁴. The high specificity of these molecular assays is directly related to the annealing specificity of the primer/probe to the genomic region. The World Health Organization (WHO) has recommended 26 primers and probes to be used in this type of diagnosis⁵.

Objective: l-Asparaginase (ASNase) is an enzyme used in the treatment of acute lymphoblastic leukemia (ALL). As the therapeutic ASNases has bacterial origin, severe side effects are associated with its use, among them hypersensitivity and inactivation of the enzyme. In this context, the objective of this work was to produce a recombinant ASNase of bacterial origin in human cells in order to determine the presence and consequences of potential post-translational modifications on the enzyme. Results: Recombinant ASNase was expressed in human cells with a molecular weight of 60 kDa, larger than in Escherichia coli, which is 35 kDa. N-glycosylation analysis demonstrated that the increased molecular weight resulted from the addition of glycans to the protein by mammalian cells. The glycosylated ASNase presented in vitro activity at physiological pH and temperature. Given that glycosylation can act to reduce antigenicity by masking protein epitopes, our data may contribute to the development of an alternative ASNase in the treatment of ALL in patients who demonstrate side effects to currently marketed enzymes.

The aim of this work was to evaluate the effect of the Rolipram during the maturation of bovine oocytes and gene expression of embryos produced in vitro. Bovine ovaries were collected in slaughterhouse. The COCs were selected and divided into 5 groups: Control 0 time; Control: IVM for 24 hours; Rolipram treatments with IVM blocking for 24 hours in maturation medium containing (100, 150 and 200µM). After 24 hours all groups were reseated in IVM for another 24 hours. Subsequently COCs were subjected to the same IVM system and fertilized, being checked for cleavage post fertilization and for blastocyst. In addition, performed expression of the following genes: Mater, BMP15 and Bax. No difference was found in gene expression. Of oocytes evaluated shortly after follicular aspiration, 79.00% were in GV, GVBD, MI, while 13.40%, were in MII and 7.60%, D/NI. Significant difference was observed in different concentrations (T100, T200 and T150µM) in oocytes that have reached the MII phase compared to control treatments (P= 0.003). Differences were observed in cleavage rate (P< 0.05) between T150 and T200 when compared to the C/24 Group. A high difference was observed on blastocyst rate (P< 0.001) among treatments compared to the control group.