http://hdl.handle.net/11422/24 Wed, 08 Apr 2026 22:50:32 GMT 2026-04-08T22:50:32Z http://hdl.handle.net/11422/28996 Title: Avaliação da atividade protetora de antioxidantes sintéticos contra danos no dna em Saccharomyces cerevisiae Author(s)/Inventor(s): Mattos, Larissa Maura de Melo Advisor: Pereira, Marcos Dias Abstract: DNA damage caused by chemical and physical agents compromises cellular integrity, potentially leading to mutagenesis, mitochondrial dysfunction, and cell death. Antioxidant complexes have been studied for their ability to protect cells against oxidative stress and genotoxic damage. This thesis evaluated the protective activity of complexes containing iron, manganese, and copper, coordinated to the H2BPClNOL ligand, against damage induced by methyl methanesulfonate (MMS) and UVA and UVB radiation in Saccharomyces cerevisiae. For MMS analysis, three strains of S. cerevisiae were used: a wild-type strain (BY4741) and two DNA repair-deficient strains, rad9Δ and rad54Δ. For UVA and UVB radiation analysis, two strains were used: a wildtype strain (FF18733) and a strain deleted for a yeast NADPH oxidase, yno1Δ. To evaluate cellular protection, survival, mutagenesis, and mitochondrial dysfunction assays were performed using spot test or plating methodologies, in addition to intracellular oxidation assays to measure the antioxidant capacity of the complexes. Furthermore, proteomic analysis was conducted to identify proteins differentially expressed in cells treated with the complexes for 1 hour. The results demonstrated that all complexes significantly increased the survival of the strains exposed to MMS, UVA, and UVB, indicating a protective action independent of the specific DNA repair pathway. Mitochondrial dysfunction assays revealed a significant reduction in mitochondrial damage caused by the stress agents, while induced mutagenesis was attenuated. Intracellular oxidation analysis showed that exposure to stress agents increased reactive oxygen species levels, whereas the complexes reduced this oxidative stress, suggesting a direct antioxidant mechanism. Proteomic analysis identified proteins differentially expressed in response to treatments, including proteins involved in DNA repair, oxidative stress response, and maintenance of cellular integrity. Proteins related to antioxidant defense and molecular chaperones were consistently regulated by the complexes, corroborating the functional results observed in survival and mutagenesis assays. Overall, the results indicate that complexes containing the H2BPClNOL ligand exert a multifactorial protective action in S. cerevisiae, mitigating oxidative stress and preserving DNA integrity and mitochondrial function. Proteomic analysis supports the hypothesis that these complexes modulate cellular pathways related to DNA repair and antioxidant defense, providing insights into cellular protection mechanisms that may be explored in future studies. Therefore, this work contributes to understanding the protective effects of metallic antioxidant complexes and highlights their potential as tools to study the interaction between oxidative stress, DNA damage, and adaptive cellular responses in eukaryotic models. These findings also reinforce the relevance of using S. cerevisiae as an experimental model to evaluate antioxidant complexes and investigate cellular repair and protection mechanisms. Publisher: Universidade Federal do Rio de Janeiro Type: Tese Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/11422/28996 2025-01-01T00:00:00Z http://hdl.handle.net/11422/28993 Title: Elucidação estrutural da manana cristalina isolada do endosperma de sementes de açaí (Euterpe oleracea mart.) Author(s)/Inventor(s): Miguez, Ingrid Santos Advisor: Silva, Ayla Sant’Ana da Abstract: Açaí seeds (E. oleracea Mart.) are a byproduct of pulp production, with 50% of their total dry weight consisting of mannan. The depolymerization of mannan releases mannose and mannan-oligosaccharides. However, açaí seeds are recalcitrant to deconstruction by enzymatic action. Understanding the structure of this polysaccharide can help identify the limitations of its processing, with the aim of increasing the efficiency of bioconversion. For this purpose, an insoluble fraction was isolated from the seeds and evaluated by solution nuclear magnetic resonance (NMR). The 1H/13C HSQC spectra were attributed to a β-Dmannan, and the 1H/13C HMBC spectra confirmed the presence of a β(1→4) glycosidic linkage between the Manp units through cross-peaks at 4.73/78.0 ppm (H-1/C-4) and 3.88/101.8 ppm (H-4/C-1). However, due to the difficulty of solubilizing the sample, the isolated fraction was also evaluated by solid-state NMR. The results confirmed it to be a linear mannan, with the presence of crystalline polymorphs I and II, detected by signals corresponding to carbon 5 of both forms at 70.2 and 70.9 ppm, respectively. The crystallinity of this fraction was assessed by X-ray diffraction (XRD), which showed peaks at 2θ values of 16°, 18°, 20°, 24°, and 26°, corresponding to planes 110, 111, 200, 210, and 211, respectively. This profile is similar to that of a crystalline form of type I mannan, which predominated in the sample. However, the XRD profile also revealed a peak at 22.5°, characteristic of the 200 crystalline plane of cellulose Iβ, indicating possible sample contamination. The contamination was corroborated by the thermal degradation behavior of the sample and compositional analysis, which indicated a 78:19:2 ratio of Man:Glc:Gal. Consequently, the sample was reevaluated using solution NMR equipped with a cryoprobe to increase the sensitivity of the method. However, it was not possible to observe the 1H/13C signal corresponding to the anomeric position of the Gal-(1→ residue, making further optimization of the method necessary to confirm the chain branching. To overcome contamination issues, the mannan from açaí seeds was also analyzed in situ through immunolocalization. The analysis not only allowed for the identification of linear mannan in the mature endosperm cell wall but also enabled the monitoring of the transformation of galactomannan into linear mannan throughout fruit development. Thus, the structure of mannan in açaí seeds was identified for the first time. The results obtained can guide strategies to reduce the crystallinity of mannan and select more suitable enzymes for the hydrolysis of this recalcitrant biomass. Publisher: Universidade Federal do Rio de Janeiro Type: Tese Thu, 05 Sep 2024 00:00:00 GMT http://hdl.handle.net/11422/28993 2024-09-05T00:00:00Z http://hdl.handle.net/11422/28988 Title: Expressão de anticorpos monoclonais anti-SARS-CoV2 em células CHO Author(s)/Inventor(s): Marsili, Federico Francisco Advisor: Castilho, Leda dos Reis Abstract: Monoclonal antibodies (mAbs) currently represent the most relevant group of biological products in the biopharmaceutical market. Because these molecules are composed of two different polypeptide units — two light chains (LC) and two heavy chains (HC) — several strategies can be implemented to enable their proper expression in heterologous systems. Among these strategies, the use of monocistronic vectors (each containing the coding sequence for either LC or HC) and polycistronic vectors (in which two or more genes or cistrons are expressed from a single mRNA molecule) are well described, being the lader commonly based on internal ribosome entry site (IRES) elements. In this context, two mAbs with neutralizing activity against SARS-CoV-2, previously isolated by other research groups, were selected as model molecules to evaluate the application of a modular expression vector system based on native and adenuated versions of the IRES element from encephalomyocarditis virus (EMCV). The vector design enabled the generation of different types of constructs — mono-, bi-, or tricistronic —, alternating the positions of the LC- and HC-encoding genes relative to the EMCV IRES element. In the tricistronic version, an adenuated version of the EMCV IRES element was introduced upstream of a gene that assists in the detection (encoding green fluorescent protein) or in the amplification of gene expression (encoding the dihydrofolate reductase enzyme). For both mAbs, transient transfection experiments performed in Chinese hamster ovary (CHO) cells of the ExpiCHO™ playorm, showed higher production levels associated with the use of vectors containing the LC gene in the first cistron (for both bicistronic and tricistronic constructs). mAbs obtained by transient transfections using bicistronic vectors with the LC gene in the first cistron were purified by Protein A affinity chromatography and subsequently subjected to physicochemical, structural, and functional characterization. The study allowed the confirmation of the amino acid sequences (by nanoRPLC/MS-MS); the evaluation of secondary, tertiary, and quaternary protein structures (by circular dichroism, intrinsic fluorescence, and SEC-HPLC and TEM, respectively); the determination of electrophoretic migration profiles (by SDS-PAGE and western blot), size distribution (by SEC-HPLC), and N-glycosylation profiles (by HILIC-HPLC and LC/MS). In addition, the ability of the mAbs to bind to different variants of the SARS-CoV-2 spike glycoprotein (by spot blot and LSPR) and to neutralize different viral strains (by in vitro neutralization assays) was also evaluated. Furthermore, one of the mAbs was selected as a candidate molecule for the development, through stable transfection, of constitutively producing cell lines based on the ExpiCHO™ playorm. Two different lipofection protocols were compared, using the cationic lipids Lipofectamine™ 3000 and ExpiFectamine™. Taken together, the results demonstrate that bicistronic vectors based on the EMCV IRES element and containing the LC gene in the first cistron represent an efficient strategy for mAb production in CHO cells, enabling the generation of antibodies that are properly characterized from both structural and functional perspectives, and that the different stable transfection protocols evaluated in ExpiCHO™ cells led to similar volumetric yields (4,1 and 6,4 mg/L). Publisher: Universidade Federal do Rio de Janeiro Type: Tese Mon, 15 Dec 2025 00:00:00 GMT http://hdl.handle.net/11422/28988 2025-12-15T00:00:00Z http://hdl.handle.net/11422/28930 Title: Estudo do estresse com metilglioxal nas interações TDP-43 - SOD1 em célula H4 como modelo para a esclerose lateral amiotrófica Author(s)/Inventor(s): Ribeiro, Gabriela Delaqua Advisor: Eleutherio, Elis Cristina Araujo Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the loss of motor neurons. Proteopathy is a pathological hallmark of the disease. In approximately 97% of ALS cases, aggregates of TDP-43 are observed in the cytoplasm of motor neurons undergoing neurodegeneration. Additionally, misfolded SOD1 is found in the neurons of patients with both sporadic and familial cases of the disease. TDP-43 is a protein associated with the regulation of transcription, translation, and RNA processing, and is predominantly located in the nucleus, while SOD1 is an antioxidant enzyme present in the cytoplasm, nucleus, and intermembrane space of mitochondria. Previous studies from the group have shown that glycated SOD1 leads to increased phosphorylation of TDP-43, considered a disease biomarker. Furthermore, the literature suggests a potential relationship between SOD1 and calcineurin, a phosphatase responsible for the dephosphorylation of TDP-43. In this context, the objective of the study was to evaluate whether TDP-43 could interact with SOD1 and the effect of methylglyoxal (MGO) and the SOD1G93A mutation, found in ALS patients, on this interaction. Neuroglial (H4) cells were used as a model. The MGO treatment resulted in decreased cell viability, reduced SOD1 activity, and lowered phospho-TDP-43 levels. Cells expressing SOD1G93A showed lower levels of SOD1 activity and phospho-TDP-43 compared to those expressing SOD1WT under control conditions without MGO. The TDP-43–SOD1 interaction was observed using the BiFC (bimolecular fluorescence complementation) system, evidenced by the reconstitution of the Venus protein and fluorescence emission during the physical interaction of the proteins. According to the results, both SOD1WT and the G93A mutant interact with TDP-43. This interaction occurs in various cellular compartments, with a higher proportion of cells exhibiting TDP-43 interacting with SOD1G93A in the cytoplasm. MGO did not alter the cellular compartments of the TDP-43–SOD1WT interaction, only leading to an increase in cytoplasmic inclusions at the 0.4 mM MGO concentration. The observed inclusions did not colocalize with stress granules. Treatment with Cyclosporin A, an inhibitor of the phosphatase calcineurin, reduced the proportion of cells with TDP-43–SOD1WT inclusions and those with TDP-43–SOD1G93A interaction in the cytoplasm. In conclusion, TDP-43 phosphorylation plays an important homeostatic role, and its imbalance leads to altered compartmentalization of TDP-43 and inclusion formation. SOD1 is crucial in maintaining the balance between phosphorylation and dephosphorylation due to its influence on calcineurin activity. This effect is not observed in the G93A mutant, which makes these cells more sensitive to changes in TDP-43 localization, an important marker of ALS. Publisher: Universidade Federal do Rio de Janeiro Type: Dissertação Wed, 01 Jan 2025 00:00:00 GMT http://hdl.handle.net/11422/28930 2025-01-01T00:00:00Z