Teses e dissertaçõeshttps://repositorio.ufscar.br/handle/ufscar/53582024-03-19T04:55:25Z2024-03-19T04:55:25ZEfeitos toxicológicos da nanopartícula de dióxido de titânio (TiO2) utilizando modelos experimentais in vitro e in vivoFattori, Ana Carolina Maragnohttps://repositorio.ufscar.br/handle/ufscar/195612024-02-29T17:20:02Z2023-11-30T00:00:00ZEfeitos toxicológicos da nanopartícula de dióxido de titânio (TiO2) utilizando modelos experimentais in vitro e in vivo
Fattori, Ana Carolina Maragno
The advent of nanotechnology and its applications in diverse areas is due to the increase in the physical and chemical properties of nanoparticles because of their nanometric scale. However, these new characteristics can trigger biological effects that harm health and the environment, highlighting the need to assess the safety of using these nanomaterials. Therefore, this study analyzed the toxicological effects of titanium dioxide nanoparticles (TiO2 NP) functionalized with sodium carboxylate (-COO-Na+), of interest to the oil industry, using in vitro and in vivo models. The physicochemical characterization of the TiO2 NP was carried out using Dynamic Light Scattering (DLS), determining its hydrodynamic size, zeta potential and polydispersity index (PdI), Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) and High-Resolution Scanning Electron Microscopy (SEM-FEG). For the in vitro model, the LA-9 fibroblast lineage was used and cytotoxicity was evaluated for the concentrations of 250, 150 and 50 μg/mL using the cell viability assay (MTT) and cell morphology analysis after exposure to TiO2 NP for 24, 48 and 72 hours. After exposure to TiO2 NP for 24 hours, intracellular production of Reactive Oxygen Species (ROS), production of cytokines IL-6 and TNF in the cell supernatant by ELISA and cell death by flow cytometry were also evaluated, as well as clonogenic survival 7 days after exposure. In the in vivo model, Balb/c mice were exposed intranasally to TiO2 NP (500, 250, 100 and 50 μg/animal) in 4 doses over 14 days. Throughout the exposure period, the animals' feed and water intake and weight variation were assessed. Subsequently, leukocyte levels were analyzed in the blood, bronchoalveolar lavage (BAL) and peritoneal cavity lavage (PCL), quantification of cytokines (INF-ɤ, TNF, IL-6 and IL-10) in plasma and BAL and IgE antibody in BAL by ELISA, evaluation of liver function (TGO/TGP), weighing of the lungs and histological analysis of the organs (brain, lungs, heart, liver, spleen and kidneys). Our results showed that NP TiO2 is functionalized with sodium carboxylate ligands and suggests that it has an anatase crystalline form, as well as heterogeneous dispersion and a hydrodynamic size of around 3.5 nm in water and 2.96 nm in PBS, increasing to 7.62 nm in DMEM medium due to the agglomeration process. For the LA-9 fibroblast cell line, there was a reduction in cell viability (250 and 150 μg/mL) dose and time-dependent as well as oxidative stress and cell death by apoptosis (150 μg/mL). For the 150 μg/mL concentration there was a possible cell recovery after the NP was removed. In the in vivo model, NP generated histopathological changes in the brain, lungs, liver and kidneys of Balb/c mice after intranasal exposure with 4 doses over a period of 14 days. No changes were observed in the other parameters evaluated, with the exception of an increase in TGO at a concentration of 250 μg. The results of this study showed that NP TiO2 functionalized with sodium carboxylate has cytotoxicity for the LA-9 fibroblast line and toxicity with histopathological alterations for the brain, lung, liver and kidneys in Balb/c mice, contributing to knowledge about the effects of this NP TiO2 in in vitro and in vivo models. It is also interesting to analyze long-term exposure in order to determine the safety of using this nanomaterial.
2023-11-30T00:00:00ZImunogenicidade de proteínas recombinantes de Erysipelothrix rhusiopathiae: proteção em um modelo murinoGodoy, Naiane Limahttps://repositorio.ufscar.br/handle/ufscar/195292024-02-28T12:35:39Z2023-12-08T00:00:00ZImunogenicidade de proteínas recombinantes de Erysipelothrix rhusiopathiae: proteção em um modelo murino
Godoy, Naiane Lima
Erysipelothrix rhusiopathiae is a Gram-positive bacillus that causes swine erysipelas, which leads to the loss and poor development of pigs. Existing vaccines for immunization against erysipelas are made up of killed or attenuated bacteria and, according to reports in the literature, have failed to provide protection, leading to the search for other components that could form a new vaccine against this disease. The surface protein SpaA of E. rhusiopathiae alredy has been reported to induce a host immune response against the bacteria, and hsp70 DnaK has been shown to be an antigenic protein of the bacteria. This work evaluated the immunogenicity and protection induced by the recombinant proteins SpaA and DnaK from E. rhusiopathiae in a murine model. The model was designed in 6 groups of mice that received two doses (subcutaneous and intramuscular) of 40 µg and 100 µg of recombinant proteins. The proteins were evaluated separately and together, compared with a commercial vaccine and with the no-vaccination condition. The proteins were evaluated separately and together, compared with a commercial vaccine and the without vaccination condition. 21 days after the last dose, mice were challenged with a virulent strain of E. rhusiopathiae and serum was collected to assess antibodies, peripheral blood cells were counted, and spleen and kidney tissues were analyzed for the presence of E. rhusiopathiae by colony count. Finally, two survival curves were performed, evaluating the protection induced by the proteins. The results showed that IgG antibodies increased in the serum of animals inoculated with the proteins, which also had bacterial proliferation reduced in the spleens and kidneys evaluated, and presented a significant delay in the symptoms of the disease in this immunization model. These results suggest that the DnaK and SpaA proteins from E. rhusiopathiae have immunogenic potential and can modulate the development of the disease, as assessed in the murine model.
2023-12-08T00:00:00ZCaracterização fenotípica, genômica e transcriptômica de parasitos Crithidia sp. LVH60a obtidos de pacientes diagnosticados com Leishmaniose VisceralRogerio, Luana Aparecidahttps://repositorio.ufscar.br/handle/ufscar/190262023-12-18T12:26:16Z2023-11-09T00:00:00ZCaracterização fenotípica, genômica e transcriptômica de parasitos Crithidia sp. LVH60a obtidos de pacientes diagnosticados com Leishmaniose Visceral
Rogerio, Luana Aparecida
Leishmaniasis is a group of neglected diseases that can be caused by over 20 species of protozoa from genus Leishmania. Since 1980, monoxenous trypanosomatids have been found in cutaneous and visceral leishmaniasis cases in both immunocompetent and immunocompromised patients. In Brazil, parasites of Crithidia genus were first reported in 2019 in a fatal case of human visceral leishmaniasis in Sergipe (SE), referred to as Crithidia sp. LVH60A. To characterize clinical isolates of Crithidia sp. LVH60A parasites obtained from a relapsing patient from the same region, this study aimed to characterize phenotypically and perform genomic and transcriptomic analyzes of these clinical isolates. In this regard, parasite cultures were analyzed for growth curves, morphological features, in vitro infectivity and in silico identification of virulence-related proteins in Crithidia sp. LVH60A genome. Genomic DNA was sequenced, assembled, and compared to other trypanosomatid species. Additionally, we assessed the difference in gene expression of parasites and human macrophages infected with different species of Leishmania and Crithidia using messenger RNA sequencing (mRNA-seq), employing a dual RNA-seq approach. Phenotypic analyses demonstrated that the Crithidia sp. LVH60A parasites were capable of replicate at temperatures of 25 °C and 35 °C, surviving and multiplying within different macrophage lineages. Morphological analyses revealed that the cellular body of Crithidia sp. LVH60A is rounded, possesses shorter flagella than L. infantum, and presents some differences when compared to the Crithidia fasciculata TCC039E strain. Genomic analyses showed that the clinical isolates from 2017 had over 98% nucleotide identity with the LVH60a C1 strain (Crithidia sp. LVH60A). Genome alignments of Crithidia sp. LVH60A and C. fasciculata strains showed an average nucleotide identity ranging from 92.7% to 94.8%, depending on the software used. Chromosomal rearrangements of Crithidia sp. LVH60A was also observed and validated when compared to C. fasciculata CfCl reference genome. The mRNA-seq results showed that infection with different parasite strains did not significantly alter the transcriptional expression of macrophages; however, slight changes were observed shortly after the infection. However, there was a clear difference in the expression of the trypanosomatid transcripts. Some differentially expressed transcripts are shared by strains of Leishmania spp. and Crithidia sp. LVH60A. These transcripts encode proteins that mediate responses to stress, nutrition, and host immune response evasion. Our results suggest that the Crithidia sp. LVH60A may be a new species within the Crithidia genus; however, more isolates of Crithidia sp. LVH60A need to be analyzed with a focus on population genomics to confirm this evidence.
2023-11-09T00:00:00ZFerramentas moleculares aplicadas ao estudo de parasitas emergentes em Leishmaniose visceralTakamiya, Nayore Tamiehttps://repositorio.ufscar.br/handle/ufscar/189202023-11-24T18:41:55Z2023-09-27T00:00:00ZFerramentas moleculares aplicadas ao estudo de parasitas emergentes em Leishmaniose visceral
Takamiya, Nayore Tamie
Leishmaniases are neglected tropical diseases considered important to public health. Visceral Leishmaniasis (VL) is the most severe form of the disease, which affects organs such as spleen, bone marrow, liver and lymph nodes. It is caused by Leishmania infantum in Brazil. Advances in Molecular Biology tools have provided alternatives for diagnostics at research reference centers. Studies have demonstrated the presence of monoxenous trypanosomatids (Leptomas spp. and Crithidia spp.) in the clinical spectrum of Leishmaniasis. The clinical implications of these trypanosomatid species in Leishmaniasis are unknown, and the impact of these infections has rarely been studied. Further research is necessary to identify and investigate these species. Therefore, this work presented two goals to advance knowledge about this emerging VL parasites. The first one was to identify new species-specific genes able of discriminating between L. infantum and Crithidia sp. in clinical and experimental samples. The secod one was to obtain genetically modified strains of parasites expressing fluorescent reporter genes through genetic engineering and cell transfection techniques, such as the green fluorescent protein (GFP) gene in Crithidia sp. LVH60A (strain LVH60a_C1) and mCherry in the HUUFS14 strain of L. infantum. Primers designed to identify L. infantum (LinJ31seq and LinJ31_2420) and Crithidia sp. (Crid2.1seq, LVH60_Tig001, and Catalase-LVH60_12060_1F) showed a good performance of detection. These primers have been extensively validated, both for species identification (qualitatively) and for estimation of parasite load (quantitatively) in experimental and clinical VL samples, covering a variety of vertebrate hosts. Through molecular screening and analysis of 62 clinical isolates from VL patients using these species-specific genes, it was possible to identify 51 parasite cultures with positive PCR results for Crithidia sp. Interestingly, qPCR assays indicated co-infection of L. infantum with Crithidia sp. LVH60A in two new cases of VL in Sergipe. Furthermore, by re-evaluating clinical samples from a case of VL from Sergipe published in 2019, it was found that the patient was co-infected with these two species of trypanosomatids. With the cell transfection experiments, it was possible to transform the HUUFS14 strain (L. infantum) expressing the fluorescent gene mCherry, presenting phenotypic characteristics similar to those of the wild-type strain and keeping infective capacity in in vitro infection assays. The transfection protocols tested in this study were inefficient for the transformation of Crithidia sp. LVH60A, highlighting the peculiarities of the parasite. Overall, this study was important in establishing new molecular targets that can be used to improve the diagnosis of VL. Furthermore, such targets show promise for future investigations, aiming to deepen our understanding of the role of monoxenic trypanosomatids in VL pathology and/or as potential emerging parasites.
2023-09-27T00:00:00Z