Mestrado em Biotecnologia
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- ItemClivagem in vitro do gene COL1A1 por CRISPR/Cas9: fundamentos para a geração de modelos experimentais(Universidade Federal do Espírito Santo, 2025-12-18) Melo, Daniel Moreira; Paula, Flavia de ; https://orcid.org/0000-0001-8679-2982; http://lattes.cnpq.br/7913201450663683; https://orcid.org/0009-0006-2579-1189 ; http://lattes.cnpq.br/4452821162268537; Santos, Eldamaria de Vargas Wolfgramm dos ; https://orcid.org/0000-0003-3815-0760; http://lattes.cnpq.br/4688343262832362; Nishimura, Agnes Lumi ; https://orcid.org/0000-0001-5295-797X; http://lattes.cnpq.br/4422870765994649Gene editing mediated by the CRISPR/Cas9 system has become a central element of modern biotechnology, enabling genome modification in a rapid, accessible and highly precise manner. Among its applications, the generation of cellular models of genetic diseases is a key area, in which a critical step is the prior evaluation of the cleavage efficiency of Cas9/gRNA complexes before their use in living cells, functioning as an initial filter to select guide RNAs with higher functional potential. Among clinically relevant genes used as experimental models, COL1A1 stands out due to its essential role in type I collagen formation and because it harbors most of the mutations associated with Osteogenesis Imperfecta, a hereditary disease characterized by bone fragility and a broad clinical spectrum. The in vitro cleavage efficiency of the CRISPR/Cas9 system targeting the COL1A1 gene was investigated using DNA from five human cell lines of different origins. Guide RNAs were designed and selected based on computational predictions of efficiency and specificity, and the fragment corresponding to exon 2 and its respective guide RNA was used in the cleavage assays. The analyses revealed intermediate and consistent efficiencies among the tested cell lines, ranging from approximately 31% to 47%. The results demonstrate that the evaluated guide exhibits reproducible activity in different genomic contexts and support the use of this workflow as a preliminary step in the generation of genetically edited cellular models. Overall, the data reinforce the importance of in vitro validation prior to cellular editing and provide an experimental basis for future studies aimed at modeling COL1A1 mutations and investigating the molecular mechanisms of Osteogenesis Imperfecta.
- ItemFatores genéticos de risco e proteção para sequelas neurológicas da COVID-19(Universidade Federal do Espírito Santo, 2025-08-04) Guaitolini, Yasmin Moreto; Meira, Débora Dummer ; https://orcid.org/0000-0002-6092-2459; http://lattes.cnpq.br/7199119599752978; Louro, Iuri Drumond ; https://orcid.org/0000-0001-5160-9615; http://lattes.cnpq.br/3817361438227180; https://orcid.org/0009-0007-6500-3092; http://lattes.cnpq.br/8081444745164015; Paula, Flavia de ; https://orcid.org/0000-0001-8679-2982; http://lattes.cnpq.br/7913201450663683; Carvalho, Elizeu Fagundes de ; https://orcid.org/0000-0003-4620-7253; http://lattes.cnpq.br/2742420738858309COVID-19, caused by SARS-CoV-2, has triggered a global crisis with significant health, social, and economic impacts. Although most infected individuals recover, some develop persistent sequelae known as post-COVID conditions, including neurological manifestations affecting the peripheral nervous system (PNS). Recent evidence suggests that host genetics may influence the susceptibility and severity of these sequelae, highlighting the importance of investigating genetic biomarkers associated with their occurrence. This is a case-control study aiming to identify genetic variants linked to the development of PNS sequelae following COVID-19, using whole-exome sequencing (WES) data. The cohort comprises 312 individuals without a complete vaccination scheme prior to infection, including 161 with sequelae (case group) and 151 without sequelae (control group). Clinical, sociodemographic, and genetic characteristics were analyzed. For genetic risk prediction, a machine learning (ML) model was implemented, testing different classifiers. The logistic regression (LR) model showed the best performance (AUC-ROC = 0.90, accuracy = 82%, and F1-score = 0.83), highlighting 20 SNPs most influential in predicting the risk of neurological sequelae. Analyses predominantly revealed pathways related to immune regulation, with the HLA-A (Antigen Peptide Transporter) gene playing a prominent role in this context. The PAQR5 gene (Progestin and AdipoQ Receptor Family Member 5), associated with steroid hormone signaling, was also identified. Additionally, other genes with undefined or poorly characterized functions, such as NPIPB15 (Nuclear Pore Complex Interacting Protein Family Member B15), possibly involved in nuclear transport, were observed. These findings suggest that immune response, inflammation, and alterations in lipid and hormonal metabolism may play a relevant role in the predisposition to neurological sequelae. The results obtained thus far provide important evidence on the genetic basis of these sequelae, contributing to the identification of susceptibility biomarkers and potential therapeutic targets, which may support advances, particularly in the clinical management of post-COVID-19 conditions
- ItemVariantes do gene NOTCH1 como biomarcadores para transtornos neurocognitivos(Universidade Federal do Espírito Santo, 2025-03-26) Rodrigues, Alda dos Santos; Paula, Flávia; https://orcid.org/0000-0001-8679-2982; http://lattes.cnpq.br/7913201450663683; Errera, Flávia Imbroisi Valle; https://orcid.org/0000-0002-8069-6372; http://lattes.cnpq.br/9337327437538048; https://orcid.org/0000-0002-8069-6372; http://lattes.cnpq.br/5759084443362110; Santos, Eldamaria de Vargas Wolfgramm dos ; https://orcid.org/0000-0003-3815-0760; http://lattes.cnpq.br/4688343262832362; Barcelos, Estevão Carlos Silva; https://orcid.org/0000-0001-7076-6165; http://lattes.cnpq.br/6899125943013073Neurocognitive disorder (NCD) can be mild (TNCL) when it does not impact independence in activities of daily living or major (TNCM), also known as dementia, is clinically heterogeneous and has been associated with different risk factors, such as metabolic diseases (diabetes, obesity), stroke, tobacco and alcohol use, among others. Genetic factors have been investigated, but are not yet fully known. Thus, the search for biomarkers that can be used in diagnosis, risk screening, prognosis and response to treatment is very important. In this context, the NOTCH1 gene is expressed in several tissues, mainly in the cerebral cortex, hippocampus and cerebellum, hematopoietic stem cells, heart and blood vessels, and is associated with 118 phenotypes. NOTCH1 encodes a protein homologous to the neurogenic NOTCH locus in humans (Homo sapiens), important for glucose homeostasis, lipid metabolism, cell growth and survival. The NOTCH signaling pathway is very important in neurogenesis and maintenance of adequate neuronal function in the human brain and has been implicated in NCD//dementia, including Alzheimer's disease (AD). Recent evidence points to a shared genetic architecture between metabolic alterations, NOTCH1 and NCD. NOTCH1 is expressed in the mature human hippocampus, and shows increased immunoreactivity in AD, and in cases with tau-positive Pick bodies. The absence of NOTCH1 is capable of affecting a cascade of secondary messengers associated with plasticity and spatial learning, and with reduced volume of brain regions, being associated with AD and, therefore, with the risk of NCD. Thus, the hypothesis tested in this work is that variants in this gene are associated with NCD. The objective is to verify whether variants in NOTCH1 are associated with NCD. Participants in the PAHO study "Health, Well-being and Aging" (SABE) from São Paulo, with complete data (N=1103), were classified for NCD using the score obtained in the Mini Mental State Examination (MMSE). Participants who scored below the MMSE cutoff (13 points) were considered cases [carriers of NCD (N=152)] and the remaining participants (951), with a score of 13 or higher, were used as controls. Clinical data were obtained from a standardized questionnaire and genetic data from whole-genome sequencing to identify genetic variants. Genotypes for the variants and their respective frequencies were obtained from the Brazilian Online Mutation Archive (ABraOM). Females comprised the majority of the population, corresponding to 64.09% (N=707). The prevalence of NCD was 13.78% (152 patients). The variants rs10870087, rs2990585, rs7874114 and rs7864342 associated with NCD were identified in DNA regulatory regions, mainly in active enhancers, suggesting their role in the regulation of gene expression, especially in brain tissues. The rs2990585 and rs7874114 variants have epigenetic marks (H3K27ac and H3K4me1), indicating involvement in the activation of genes related to synaptic plasticity and neurodevelopment. rs2990585 with a high probability of regulatory function (1f), binding to the CEBPA gene, involved in cell differentiation and inflammatory response. Enhancer 2 of this variant is active in several brain regions, such as the midbrain and frontal cortex. Furthermore, the rs10870087, rs7864342 and rs7874114 variants demonstrated eQTL activity for CYSRT1 in breast tissue, while rs2990585 presented eQTLs in genes such as NOTCH1 (blood and tibial artery) and NRARP (lymphocytes). These findings suggest that variants in NOTCH1 have an impact on gene regulation and are promising candidates for use as biomarkers of NCD. However, functional studies and studies in other populations are needed for generalization and application of these results
- ItemFluorescência da clorofila a como ferramenta indicadora de estresse mineral em Schinus terebinthifolia Raddi(Universidade Federal do Espírito Santo, 2025-03-31) Dalla, Rhaira Rodrigues; Guimarães, Marco Cesar Cunegundes; https://orcid.org/0000-0003-2146-0180; http://lattes.cnpq.br/0261991057482057; https://orcid.org/; Errera, Flavia Imbroisi Valle; https://orcid.org/0000-0002-8069-6372; http://lattes.cnpq.br/9337327437538048; Medeiros, Marcos Barros de; https://orcid.org/0000-0002-1633-3227; http://lattes.cnpq.br/6453244305834445The aim of this study was to evaluate the effectiveness of chlorophyll a fluorescence as a diagnostic tool to identify and monitor abiotic stress in Schinus terebinthifolia Raddi, which occurs due to nutrient deficiency or excess, affecting its growth and metabolism. The deficiency reduces photosynthesis and development, while excess can cause toxicity. This results in chlorosis, necrosis and reduced resistance to pathogens, compromising its adaptation and regeneration. In addition, we sought to correlate changes in fluorescence indices and the modulation of proteins involved in the protection and adaptation of the photosynthetic apparatus. In vivo analyses of chlorophyll a fluorescence were performed using a portable fluorometer at four stations located in Espírito Santo (AE1, AE2, AE3 and AE4) in plants under abiotic stress conditions. These analyses allowed obtaining parameters such as maximum photosynthetic efficiency (Fv/Fm) and non-photosynthetic energy dissipation (NPQ), evaluated by OJIIP and SPAD. In addition, a search was conducted at NCBI for potential proteins to be used in the development of diagnostic tests in plants under mineral stress. The results of the study demonstrated that the exposure of Schinus terebinthifolia Raddi to different abiotic stress conditions caused marked changes in photosynthetic efficiency and a significant reduction in maximum photosynthetic efficiency (Fv/Fm) indices, indicating damage to the photosynthetic system resulting from the applied stresses. The increase in energy dissipation via NPQ evidences functional changes in the photosynthetic system that occur early in the face of mineral stress conditions suffered by the red pepper plant. Furthermore, five proteins, OHP1, OHP2, PSBS, ELIP1 and cpSRP43, were identified as potential proteins to be tested in a greenhouse using Arabidopsis thaliana as a model organism. Thus, the study showed that chlorophyll a fluorescence is a promising tool for the diagnosis of abiotic stress in Schinus terebinthifolia Raddi, allowing the early detection of imbalances in the photosynthetic apparatus. Furthermore, the proteins mentioned can be applied in later studies because they are associated with the assembly of the photosynthetic apparatus and interaction with pigments for the synthesis of chlorophyll
- ItemO papel da L-arginina sobre o sistema oxidativo em células mda-mb-231 de câncer de mama triplo negativo(Universidade Federal do Espírito Santo, 2025-03-18) João Augusto Diniz Moura; Gouvea, Sonia Alves; https://orcid.org/0000-0001-5180-471X; http://lattes.cnpq.br/7268228122543743; Meira, Débora Dummer; Sartório, Carmem LuízaL-Arginine is a semi-essential amino acid whose role in cancer has been under investigation, showing beneficial results due to its involvement in various physiological systems. Therefore, it is necessary to investigate which pathways involving this amino acid directly contribute to tumor regression, such as those related to oxidative stress. In this context, the use of the MDA-MB-231 triple-negative breast cancer cell line, known for its high aggressiveness and limited treatment options due to the lack of therapeutic targets, highlighted the effects of different concentrations of L-ARG on cell viability and the oxidative system. The treatment involved exposure to L-ARG at concentrations of 800 µg/mL, 1600 µg/mL, and 3200 µg/mL for 48 hours. A dihydroethidium (DHE) staining assay was performed to detect the increased presence of superoxide anion (O₂⁻). The AlamarBlue assay was employed to measure cell proliferation and assess cell viability. AOPP values were determined according to the protocol described by Witko-Sarsat et al. (1996) to evaluate the production of advanced oxidation protein products. Western blotting was used to determine the influence of L-arginine treatment on SOD1 protein expression in MDA-MB-231 cells across the NT, 800 µg/mL, 1600 µg/mL, and 3200 µg/mL groups. Data were analyzed using GraphPad Prism® version 8.0.2 and corrected by Tukey’s test. The main findings of the study were: (I) Reduced cell viability; (II) Increased production of superoxide anion radicals (O₂⁻) at all concentrations; (III) Elevated levels of advanced oxidation protein products; and (IV) Reduced SOD1 expression. Therefore, through the significant results that contributed to elucidating the effects of L-arginine on the oxidative system in MDA-MB-231 cells, this study opens new perspectives with potential translational implications.