Doutorado em Genética e Biologia Molecular (ICB)
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Item Desenvolvimento e caracterização de nanopartículas mistas de quitosana e lecitina contendo melatonina para o tratamento de feridas em ratos diabéticos(Universidade Federal de Goiás, 2020-08-19) Corrêa, Viviane Lopes Rocha; Leite, Liliana Borges de Menezes; http://lattes.cnpq.br/2012543423092393; Amaral, André Corrêa; http://lattes.cnpq.br/8801299423520104; Amaral, André Corrêa; Bocca, Anamélia Lorenzetti; Tedesco, Antônio Cláudio; Diniz, Danielle Guimarães Almeida; Miguel, Marina PachecoWound healing in diabetic patients remains a worldwide problem that can cause amputations and even lead to death. This work aimed to produce melatonin-loaded lecithin-chitosan nanoparticles (MEL-NP) and to evaluate a topical formulation containing these particles for healing in an in vivo animal model for diabetes. For the production of nanoparticles, an ethanolic solution containing soybean lecithin and melatonin was dropwise added to an aqueous solution of chitosan under sonication. The nanoparticles were physical-chemical characterized and evaluated in vivo for toxicity using the Galleria mellonella model and its potential for wound healing in diabetic rats. The MEL-NP were around 160 nm in size and had a zeta potential around 25 mV. The melatonin entrapment efficiency was 27%. Our results demonstrated that treatment with MEL-NP improved wound healing demonstrated by a wound closure earlier than the other treatments evaluated. An appreciated therapeutic effect was achieved by MEL-NP in the induction of fibroblasts and angiogenic proliferation. In addition, it was accompanied by an expressive collagen deposition. Taking the observed data, the MEL-NP produced could be used in the development of promising strategies for wound healing in diabetic people.Item Investigação genômica em pacientes com deficiência intelectual ou atraso global do desenvolvimento assistidos na rede pública de saúde do estado de Goiás(Universidade Federal de Goiás, 2022-02-21) D’Ávila, Ana Julia da Cunha Leite; Minasi, Lysa Bernardes; http://lattes.cnpq.br/9057708164796074; Cruz, Aparecido Divino da; http://lattes.cnpq.br/7868817504129985; Cruz, Aparecido Divino da; Silva, Claudio Carlos da; Gigonzac, Thais Cidália Vieira; Costa, Emilia Oliveira Alves; Silva, Daniela de Melo eIntellectual Disability (ID) is a complex and heterogeneous clinical condition that affects about 1 to 3% of children and adolescents worldwide, and that can be caused by a variety of environmental and/or genetic factors. There are a variety of human genetic variants already identified for ID, from small variants to larger structural variants that affect thousands to millions of nucleotides. GTG banding karyotyping and microarray chromosome analysis (CMA) are often used to identify the genetic causes of ID, especially in cases Where the clinical evaluation indicates the syndromic form of ID. The use of next generation sequencing technologies for the diagnosis of ID has made it possible to expand the detection of new mutations and new genes associated with ID. The aim of this study was to perform total exome sequencing with gene panel analysis for intellectual disability, in patients who presented karyotype without structural and/or numerical alterations and did not present pathogenic copy number variations (CNVS) in the CMA. A retrospective cohort of 369 patients of both sexes with clinical indications of DI and/or global developmental delay was evaluated in the study. Peripheral blood samples were collected to perform cytogenetic analysis and gene sequencing. GTG banding was performed for all patients following the standard protocol. CMA was performed for patients who did not present structural and/or numerical alterations in the karyotype. Genomic DNA was isolated from patients and their biological parents in order to determine the origin of the CNVs found. Cases that were not diagnosed after performing the karyotype and CMA were referred for exome sequencing with gene panel analysis for intellectual disability, which encompassed 1,252 genes. With the karyotype, it was possible to identify chromosomal alterations in 34.7% (128/369) of the patients. CMA was performed in 83 patients who had results with no alterations in karyotype (46,XX or 46,XY) with a diagnosis rate of 21.7% (18/83). Exome sequencing for target genes was performed in 19 trios of families that had negative results in previous methodologies. Gene panel analysis of exome data identified mutations in 63.1% (12/19) of cases, of which 75% (9/12) were pathogenic variants, 8.3% (1/12) probably pathogenic and in 16.7% (2/2) VUS. Of the total variants identified, 75% (9/12) were de novo mutations and 25% (3/12) had mutations inherited from healthy parents. The most prevalent variants were missense-type mutations (66.7%) followed by nonsense-type mutations 16.7%), frameshift (8.3%) and loss CNV (8.3%). With the three methodologies applied, it was possible to identify the genetic causes of ID in 42.3% (156/369) of the patients. In conclusion, our results show different methodologies that can be used for the genetic diagnosis of ID and that exome sequencing with gene panel analysis provides an efficient diagnostic strategy when combined with clinical and laboratory screening.Item Evidência do aumento de desvios mendelianos mediante análise transgeracional de mutações germinativas em população exposta à radiação ionizante de Césio-137(Universidade Federal de Goiás, 2022-08-12) Oliveira, Lorraynne Guimarães; Costa, Emília Oliveira Alves; http://lattes.cnpq.br/2332660855010816; Cruz, Aparecido Divino da; http://lattes.cnpq.br/7868817504129985; Silva, Daniela de Melo e; Silva, Claudio Carlos da; Dias, Renata de Oliveira; Leito Filho, Hugo Pereira; Cruz, Aparecido Divino daOn September 13th, 1987, the largest radiological accident in urban areas occurred in Goiânia, Goiás (Brazil), resulting in human, animal, plant and environmental exposure by Cesium-137. The mutagenic effects on the germline of people exposed to ionizing radiation are of particular concern, as the risk of inherited disorders can be increased. Single Nucleotide Variants are the most common form of human genetic variation and occur in greater abundance in a non-coding region and in recent years, several technologies have been developed to identify these variants. One of these technologies is Next-Generation Sequencing, which offers greater amounts of data, shorter sequencing times and reduced costs. The objective of this work is to establish the spectrum and frequency / rate of base de novo substitutions of germ origin from the trio's new generation sequencing data, corresponding to an F1 generation child and his biological parents, accidentally exposed to high and low doses of IR of cesium-137, contributing to knowledge about the biological effects of exposure to ionizing radiation. Considering case and control, the results of 38 parents, and an offspring of 14 children born from the exposed group and 5 children from the unexposed population were included. Exposed individuals had ~39% increase in global mean DMs compared to controls. Exposed mothers had ~44% increase in global mean DMs compared to controls. The A:T>C:G mutation was the one that showed the greatest statistically significant increase in occurrence in the offspring of exposed individuals. Transition replacement rates were higher than transversions in the offspring of cases and controls, but the difference is not statistically significant. In conclusion, with the methodology and biomarkers used, it was possible to identify the origin of mutation in the parents, as well as the type of substitution and to inform which variant was mutated, it was also possible to detect the frequency of the germline mutation in DM which made it possible to retrospectively study this population exposed to IR.Item Desenvolvimento e testes in vivo de nanopartículas de quitosana contendo insulina na cicatrização de feridas em ratos diabéticos(Universidade Federal de Goiás, 2019-04-16) Ribeiro, Maycon Carvalho; Menezes, Liliana Borges de; http://lattes.cnpq.br/2012543423092393; Amaral, André Corrêa; http://lattes.cnpq.br/8801299423520104; Menezes, Liliana Borges de; Miguel, Marina Pacheco; Mendes, Elizabeth Pereira; Souza, Taís Andrade Dias de; Silva, Luís Antônio Dantas daChitosan has been studied for its ability to accelerate healing and has been tested in the therapy of difficult-to-heal lesions, such as in diabetic patients. Insulin acts by stimulating the signaling pathway for wound healing. The objective of this work was to produce chitosan nanoparticles containing insulin for the evaluation of cicatrizant activity in diabetic rats. For the formation of the nanoparticles, the ionic gelation method was used. The nanoparticles were analyzed by diameter, potential zeta polydispersity index. The degree of deacetylation of chitosan by potentiometric was determined. For the insulin- associated nanoparticles, the mean diameter was 245.9 ± 25.46 nm and zeta potential of 39.3 ± 4.88 mV and PDI of 0.463 ± 0.01. The mean degree of deacetylation found was 72.95%. The Bradford assay revealed that the nanoparticles incorporated 97.19% ± 2.18 of insulin. To evaluate the healing, 72 Wistar rats were divided in four groups: sepigel (S), sepigel with insulin (SI), empty chitosan nanoparticles (QV) and chitosan nanoparticles containing insulin (IQ). The groups were subdivided into three subgroups (n = 6) according to the histological analysis times of the wound (3rd, 7th and 14th day). The induction of diabetes occurred through the intraperitoneal application of alloxan (120mg / kg). After confirmation of the diabetes state, the animals were anesthetized and the wounds were made with an 8.0 mm punch in the dorsal region. Macroscopic and microscopic analyzes were performed. It was possible to produce chitosan nanoparticles by the ionic gelation method, with desired diameter and zeta potential and polydispersity index. No differences were found in the rate of wound retraction among the four groups. The topical use of empty or insulin-containing chitosan nanoparticles in wound healing in diabetic rats was able to stimulate inflammatory cell proliferation and angiogenesis, followed by wound maturation. Differences in wound healing data from the group treated with insulin-containing nanoparticles and from the group treated with free insulin may be related to the high stability of theItem Análises proteômicas de cepas de Staphylococcus saprophyticus elucidam diferentes estratégias para virulência e infecção(Universidade Federal de Goiás, 2019-05-13) Silva, Karla Christina Sousa; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Marval, Márcia Giambiagi de; Fontes, Wagner; Kipnis, André; Borges, Clayton LuizStaphylococcus saprophyticus is a gram positive, coagulase negative bacteria and is one of the most common etiological agent of urinary tract infections (UTI) among sexually active young females. The infection may be led mainly by sexual intercourse. Little is known about the molecular features of S. saprophyticus infection model. However, plasticity in the proteomic profile is detected in the genus Staphylococcus and can be related with infection ability. In this study, we aimed to analyze the metabolic differences among three different pathogenic strains of S. saprophyticus (ATCC 15305, 7108 and 9325) using proteomics approaches. The proteomic data revealed variations among the strains that may lead to different responses in the context of an infection. There are differences in the carbohydrate metabolism, amino acid metabolism and pathogen defense against host defenses. Thioredoxin was one of the differentially expressed proteins among strains, thiol level assays showed similar levels of reduced thiol for ATCC15305 and 9325 strains, in contrast 7108 strain showed considerably low levels of reduced thiol levels when compared to the other analyzed strains. Urease is one of the main virulence factors of S. saprophyticus. The cytoplasmatic urease concentrations varied considerably. The 9325 strain had the lowest cytoplasmatic urease activity, however when plated on ureabased agar it showed a high urease activity, which means that probably most of the urease synthesized is exported to the extracellular milieu. Another important feature is related to the purine metabolism operon, which is highly regulated on ATCC15305 and exerts an influence upon the biofilm production in this strain. These results show that, although belonging to the same species, different S. saprophyticus strains present diverse behavior in response to different contexts that the microorganism may face in the host.Item Montagem e análise do transcritoma do mogno africano (Khaya grandifoliola C. DC.)(Universidade Federal de Goiás, 2019-07-25) Soares, Sabrina Delgado; Novaes, Evandro; http://lattes.cnpq.br/0568272239145336; Novaes, Evandro; Telles, Mariana Pires de Campos; Soares, Thannya Nascimento; Coelho, Alexandre Siqueira Guedes; Vianello, Rosana PereiraKhaya grandifoliola C. DC. is a tropical species with high value wood in international markets. Despite the high interest for reforestation and timber production, virtually nothing is known about the genetic and genomic aspects of the species. With the development of next-generation sequencing platforms (NGS), the characterization of transcriptomes can be performed on a large scale and at affordable cost. In this context, the objective of the work was to discover K. grandifoliola genes by using RNA-Seq technique and to identify those differentially expressed between the leaf and the xylem K. grandifoliola tissues. Total RNA was extracted from leaf and xylem samples of four trees planted in the experimental area of the UFG. mRNA samples were sequenced on the Illumina HiSeq 2000 platform, in paired-end mode 2 × 100 bp. Sequencing resulted in a total of 857 million sequences which, after quality control, decreased to 808 million with an average size of 24 to 85 bp. Trinity program was used to reassemble the transcriptome, obtaining 116,289 transcripts for a total of 36,271 genes. Transcripts ranged from 524 to 12,031 bp with a mean of 1,366 bp and N50 of 1,657 bp. Analyses of gene expression identified 13,626 differentially expressed genes (DEGs) (FDR <0.05) between the two tissues. Of these, 5,438 had higher expression in leaves and 8,188 in xylem. A functional enrichment analysis of Gene Ontology categories among the DEGs was performed by the Blast2GO program. This analysis identified 26 GO terms enriched among the most active DEGs in xylem. Among genes up-regulated in leaves, 48 enriched terms were found. As expected, the categories identified as enriched between DGEs point to biological processes and cellular components specific of each tissue. For example, among the most active genes in leaf tissue, terms associated with thylakoids (GO:0009579), plastids (GO:0009536) and photosynthesis (GO:0015979) were identified. On the other hand, for the xylem, terms related to carbohydrate metabolism (GO:0005975), cell wall (GO:0071554) and cytoskeletal proteins (GO:0008092) were significantly enriched. To the best of our knowledge, this is the first report of large-scale gene sequences for K. grandifoliola. Differential expression analysis allowed us to identify which genes are most active in leaf and xylem. Analysis in iPath3 identified the metabolic routes activated in leaf and xylem tissues. A BlastX search against the orange proteome (Citrus sinensis) revealed the presence of 95 DEGs annotated for the enzymes of the limonoid, a secondary metabolite with medicinal properties.Item Homeostase de ferro em Paracoccidioides spp.: novos alvos de estudo e estabelecimento de HSP30 como proteína ligante de hemoglobina(Universidade Federal de Goiás, 2021-06-28) Souza, Aparecido Ferreira de; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Soares, Célia Maria de Almeida; Schrank, Augusto; Parente, Ana Flávia Alves; Bailão, Elisa Flávia Luiz Cardoso; Bailão, Alexandre MeloUnderstanding the mechanisms that govern host-pathogen interaction is crucial for the development of new therapeutic and diagnostic approaches. Fungi of Paracoccidioides genus are the etiological agents of paracoccidioidomycosis, a systemic mycosis and, when in the host, fungi find a hostile environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe3+ chelators), use host proteins that contain Fe as metal’s source and use a non-canonical reductive pathway for assimilation of Fe. Despite all these findings, there are still gaps that need to be filled regarding the pathogen's response to Fe deprivation. In the present work, targets that still need to be studied in this context were defined, such as the establishment of the protein functions of the fungus that contain the CFEM domain and the determination of the specificity of siderophore receptors. Additionally, it was shown that the exposure of Paracoccidioides to hemoglobin promotes changes at fungal cell wall proteome level, which is illustrated by the increased ability of the fungus to interact with macrophages. This approach allowed the bioprospecting of the hemoglobin-binding protein HSP30, which is present on the cell surface and has its expression regulated positively when the fungus is exposed to hemoglobin. It has been shown that silencing of hsp30 causes decreased growth of the fungus after exposure to hemoglobin. It is also necessary to investigate the function of HSP30 as heme oxygenase. The detailed observation of those open questions can promote the expansion of knowledge about the biology of Paracoccidioides spp. and, consequently, promote the bioprospecting of new targets for differential diagnosis and/or therapy of PCM.