Mestrado em Genética e Biologia Molecular (ICB)
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Item Mesma informação, novas aplicações: revisitando primers do gene COI em aves e melhorando a identificação por DNA barcoding(Universidade Federal de Goiás, 2021-03-31) Melo, Amanda Alves de; Nunes, Rhewter; http://lattes.cnpq.br/6169806655018346; Telles, Mariana Pires de Campos; http://lattes.cnpq.br/4648436798023532; Telles, Mariana Pires de Campos; Braga, Ramilla dos Santos; Targueta, Cíntia PelegrinetiMolecular identification of species occurs through the comparison of either morphological, biochemical or genetic characteristics. All of these areas of knowledge for delimiting species are connected and should be analyzed as a complement to fill gaps of information of each area. The process of molecular identification of species through comparison of genetic information named DNA barcode consists of analyzing nucleotide sequences of a certain region to compare the differences between species. On the majority of the animals, including birds, the cytochrome c oxidase I (COI) gene is widely used for this purpose and in most cases, it shows the ideal greater variation in its sequence among species rather than within individuals of the same species, providing elucidative information about species differentiation. Choosing primers for a DNA barcode application is a crucial step because it depends on the aim of the study and on the taxonomic group of interest. Primers in silico analysis can evaluate its physicochemical properties, the number of species reached by them and then direct the choice of the best primers for the different research objectives, saving time and money resources. Therefore, this present work aimed to answer questions related to the efficiency of the available primers for the avian COI gene and to the avian mitochondrial genome sequences available in databases: i) What is the taxonomic coverage (number of species) reached by the avian COI gene primers? Are the universal primers actually capable of covering all species?; ii) Which avian orders have the most mitochondrial genome sequences available on the databases and what is its representativeness significance within the total number of species for each order?; iii) Which primers show the ideal physicochemical properties to increase the chances of successful amplification in laboratorial experiments? and; iv) Which primer sets are the most suited to guarantee full species recovery within the avian group?Item Malato sintase de Paracoccidioides brasiliensis é uma proteína ligada à superfície que se comporta como uma anchorless adesina(Universidade Federal de Goiás, 2009-05-11) Silva Neto, Benedito Rodrigues da; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Soares, Célia Maria de Almeida; Lenzi, Henrique LeonelThe pathogenic fungus Paracoccidioides brasiliensis causative of Paracoccidioidomycosis (PCM), a pulmonary mycose acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues leading to a severe form of the disease. Adhesion and invasion to host cells are essential steps involved in the internalization and dissemination of pathogens. Inside host, P. brasiliensis use the glyoxylate cycle for intracellular survival. Here, we provide evidence that malate synthase of P. brasiliensis (PbMLS) is localized on the cell wall, and is secreted. PbMLS was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By using Confocal Laser Scanning Microscopy and Western blot analysis, PbMLS was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis of mother and bud yeast cells. PbMLSr and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells A549. These observations indicated that cell wall-associated MLS of P. brasiliensis could be mediating the binding of fungal cells, thus contributing to the adhesion of fungus to host tissues and to the dissemination of infection.Item Variantes polimórficas em genes de detoxificação celular e suas relações com o desenvolvimento da esclerose lateral amiotrófica(Universidade Federal de Goiás, 2020-03-05) Santos, Kamilla de Faria; Santos, Rodrigo da Silva; http://lattes.cnpq.br/4806187026900959; Reis, Angela Adamski da Silva; http://lattes.cnpq.br/3243656364470085; Reis, Angela Adamski da Silva; Bailão, Alexandre Melo; Souza, Guilherme Rocha Lino deAmyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons, leading to progressive muscular atrophy. Studies suggest the relationship of the disease with environmental and genetic factors, and the involvement of mechanisms, such as oxidative stress, with neuronal degeneration. However, the body has several ways to promote cellular detoxification against harmful compounds. The Glutathione S-transferase (GST) family consists of multifunctional enzymes that act on cell detoxification and elimination of various substances, including oxidative stress products. Among the human cytosolic classes of the GST family, two isoenzymes are highlighted: GSTM1 and GSTT1, both encoded by genes of the same name, these genes have a total deletion polymorphism that results in the absence of enzymatic activity. Therefore, the objective of the present study was to evaluate the deletion polymorphisms in GSTM1 and GSTT1 genes and their association with the risk of developing ALS. A case-control study was conducted, including 101 patients diagnosed with ALS and 119 individuals without diagnosis of neurodegenerative diseases. Peripheral blood samples were collected from both groups and subjected to DNA extraction and subsequent genotyping. The polymorphisms were genotyped by the multiplex real - time PCR (qPCR) technique, with the definition of the null and present genotypes by analysis of the melting curves produced after the amplification. Clinical and demographic data were collected from medical records and questionnaires, including topics such as cigarette use, alcohol intake, age of diagnosis, physical activity practice, occupational history, among others. Among the groups analyzed, alcohol consumption was predominant in patients with ALS, with a significant difference between the case and control groups (p=0.01). However, there was no association of GSTM1 (p=0.85), GSTT1 (p=0.90) deletion polymorphisms and their possible genotypic combinations with the risk of developing ALS. The relationship of polymorphisms with the clinical and demographic profile of patients with the disease was also performed. In this analysis, there was a significant difference in the GSTM1-present genotype with the variables: environmental exposure and smoking (p = 0.02 and 0.03, respectively), in the GSTT1-present genotype with a history of neurodegenerative disease in the family (p=0.01), while the double genotype present also showed a significant difference with the family history of neurodegenerative disease (p=0.02). In this context, the results found in this study demonstrate the non-association of GSTM1 and GSTT1 deletion polymorphisms with the development of ALS.Item Toxicidade de nanopartículas de óxido de ferro (γ-Fe2O3) funcionalizadas com citrato ao longo do desenvolvimento inicial do zebrafish (Danio rerio)(Universidade Federal de Goiás, 2020-01-24) Pereira, Aryelle Canedo; Gonçalves, Bruno Bastos; http://lattes.cnpq.br/3194796958722741; Rocha, Thiago Lopes; http://lattes.cnpq.br/6325937100056775; Rocha, Thiago Lopes; Rodrigues, Gisele Augusto; Franchi, Leonardo PereiraIron oxide nanoparticles (NOFs) are being increasingly used in medical, environmental and technological applications. However, with its growing production and application, concerns arise about its release into the environment and its impact on human and environmental health. In this sense, the zebrafish (Danio rerio) is proving to be an excellent model system for the analysis and classification of nanomaterial toxicity. Thus, the general objective of the present study was to evaluate the toxicity of NOFs (γ-Fe2O3 NPs) functionalized with citrate throughout the initial development of zebrafish, comparing toxicity with iron ions, after static and semi-static exposure. For this purpose, the zebrafish embryos were exposed in static (without medium renewal) and semi-static (medium renewal every 24 h) exposure modes to γ-Fe2O3 NPs functionalized with citrate and iron chloride in different concentrations environmentally relevant iron (0.3, 0.6, 1.25, 2.5, 5.0 and 10 mg L-1) for 144 h, together with the control group maintained in water reconstituted in 24-well plates. The results showed that γ-Fe2O3 NPs were accumulated mainly in the chorion of the embryos and in the digestive system and liver of zebrafish larvae. Γ-Fe2O3 NPs have low toxicity compared to iron ions, indicating that the in vivo toxicity of both forms of iron is mediated by different mechanisms of action. However, NPs caused significant changes after semi-static exposure causing a decrease in heart rate, induced blood accumulation and formation of pericardial edema in zebrafish embryos, indicating its cardiotoxic effect. Regarding the exposure method, it was observed that both forms of iron induced high embryotoxicity under semi-static exposure conditions compared to static exposure, indicating that the toxicity depends on the frequency of exposure. This was the first study on the impact of the exposure condition on the toxicity of γ-Fe2O3 NPs on the development of zebrafish.Item Hibridização in situ por fluorescência: análise histórica, inovações e perspectivas(Universidade Federal de Goiás, 2022-09-05) Silva, Thainá Ferreira; Pinto, Rafael Barbosa; http://lattes.cnpq.br/9944641590654242; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056; Soares, Thannya Nascimento; Nunes, Rhewter; Machado, Raquel MouraFluorescence in situ hybridization (FISH) is a molecular cytogenetic technique that is based on the use of fluorescence-labeled probes to identify different regions in the genome. From the improvement and development of new technologies, variations of the original technique emerged making it possible to apply it to various issues, from identifying chromosomes through descriptive cytogenetics to making evolutionary inferences or conducting applied research in biotechnology and genetic improvement. Considering the great possibility of applications of the FISH technique and its variations, the present work aims to detail the methodological principles and applications, with emphasis on plants, and to investigate its use in evolutionary studies. For this, the first chapter is a manuscript for the dissemination and popularization of science that aims to discuss the various possibilities of using the FISH technique, ranging from its methodological principles to applications in different areas. From this study, it was verified that FISH can be applied to different biological materials and regions of the genome and, despite being a technique developed for more than three decades, it can generate impacts and contributions through its wide use until today. The second chapter is a systematic review, whose main objective is to summarize and critically analyze the data available in the literature on the applications of FISH for the cytogenetic study in plants, in addition to detailing and discussing the applications of this technique for evolutionary studies in plants. This study was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search was carried out in the Scopus database and the keywords were directed to capture the breadth of the use of the FISH technique aimed at the cytogenetic study of plants. In order to investigate the applications of this technique, the articles found were grouped into different categories, called “Plant Biotechnology”, “Evolutionary Studies”, “Descriptive”, “Techniques/Methodological” and “Gene Expression”. A total of 4006 articles were selected that showed a trend of growth in the number of publications between the years 1975 - 2020. China and the USA concentrated the largest number of articles and Brazil was in 8th position. The analysis of the most used keywords in the articles made it possible to trace a small history of the technique, ranging from the beginning of indirect labeling in plant species, passing through the advances of FISH and its variations, to the use of oligo-synthetic probes today. The categories “Plant Biotechnology” and “Evolutionary Studies” represented more than 60% of the total works, with emphasis on the use of GISH and FISH techniques with repetitive DNA probes and indirect labeling. The Poaceae family stood out in the studies of "Plant Biotechnology" and Leguminosae and Asteraceae were mostly found in the category of "Evolutionary Studies", “Comparative analysis” and “Systems and cytotaxonomy.” Thus, the understanding of the technique, the synthesis and measurement of data and the evolutionary path elaborated in this study allowed the theoretical basis about the versatility of the technique and its use today. demonstrate the main contributions of FISH directed to studies with plants, highlighting the regions of research concentration, variations of the technique, main types of studies and botanical families, as well as contributing to answering different evolutionary questions.Item Análise proteômica do fungo patogênico humano Fonsecaea pedrosoi submetido à temperatura do hospedeiro(Universidade Federal de Goiás, 2021-09-29) Rosa, Gabriela Danelli; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, Alexandre Melo; Lima, Patricia de Sousa; Rocha, Thiago LopesChromoblastomycosis (CBM) is a chronic subcutaneous mycosis, very common in tropical and subtropical regions and affects many men related to rural activities. Lesions of this disease can appear in five clinical forms and treatments are difficult due to the recalcitrant nature of the disease. The main causative agent of CBM is the fungus Fonsecaea pedrosoi, which is a polymorphic, melanized fungus that presents a certain phenotypic plasticity in face of temperature variations. Thermotolerance is one of the virulence factors of virulence for this pathogen, since it survives the temperature increase, like that of the host. However, no work so far has searched to characterize the adapted cellular processes. Proteins are part of the strategies used in response to a variety of stressors, such as host temperature. In this work we mapped the intracellular proteomic profile of F.pedrosoi cultivated under different temperature conditions (28 and 37 °C) for 24 hours. It was possible to identify by means of Liquid Chromatography coupled to Mass Spectrometry (Nano UPLC-MSᴱ) a total of 486 differentially expressed proteins, 101 of which were up-regulated and 385 down-regulated. It was possible to observe that F. pedrosoi seems to respond to temperature stress by repressing central carbon metabolism pathways, such as glycolysis, tricarboxylic acid cycle (TCA) and glyoxylate cycle, cell wall biosynthesis and melanin and induced amino acid degradation enzymes and antioxidant proteins.Item Marcadores microssatélites para os genomas nuclear e organelar de amburana cearensis (Allemão) A.C. Smith(Universidade Federal de Goiás, 2021-08-30) Santos, Laís Souza; Mansano, Vidal de Freitas; http://lattes.cnpq.br/5618078551561118; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056; Soares, Thannya Nascimento; Telles, Mariana Pires de Campos; Vianello, Rosana PereiraAmburana cearensis is a tree species, popularly known in Brazil as cerejeira, widely distributed in Brazil and also in Argentina, Bolivia, Paraguay and Peru. The species is distributed in a range of intense anthropic action and classified as endangered according to the International Union for the Conservation of Nature (IUCN). The economic demand for A. cearensis as a wood source and medicinal source has boosted its predatory exploitation, representing a threat to the survival of the species. In this scenario, conservation genetics is essential for generating knowledge about the genetic diversity of the species and defining appropriate strategies for its conservation and management. The general objective of this work was to design primer pairs with high potential for the development of robust and informative microsatellite markers for the nuclear, chloroplast and mitochondrial genomes of A. cearensis. Moreover, for quickness and cost savings, nuclear microsatellite primer pairs were designed for amplification in multiplex PCR. To achieve the proposed objectives, the genome of A. cearensis was partially sequenced, using the Illumina MiSeq platform. The assembly of the genomic library was performed from the DNA of an individual of the species and the genomic sequences obtained were analyzed using FastQC, Trimmomatic and DipSPAdes software. The search for microsatellite regions was performed using the QDD program. Thirty pairs of primers for nuclear microsatellite regions were designed, organized into five sets for amplification in multiplex PCR. The primer pairs designed in this study have the potential to generate new microsatellite markers with high individual discrimination capacity and transferability to other species of the genus Amburana. These markers can be applied quickly and efficiently for different types of genetic diversity studies of A. cearensis and Amburana genus.Item Identificação proteômica de alterações metabólicas em Paracoccidioides brasiliensis induzidas por derivado de chalcona(Universidade Federal de Goiás, 2021-09-30) Carvalho Júnior, Marcos Antonio Batista de; Silva, Kleber Santiago Freitas e; http://lattes.cnpq.br/3813868830071259; Pereira, Maristela; http://lattes.cnpq.br/1345781867765758; Pereira, Maristela; Curcio, Juliana Santana de; Soares, Célia Maria de AlmeidaParacoccidioidomycosis is one of the most important systemic mycoses in Latin America. The infection occurs through the inhalation of fungal propagules belonging to Paracoccidioides genus that are present in soils, being largely associated with the contamination of rural workers, who are constantly exposed to this potentially contaminated material. Even more than a century after its discovery, the treatment of this infection with the currently available antifungal arsenal still represents a challenge, due to the long treatment time required, as well as the high toxicity of the drugs used. These questions highlight the need for research to develop and characterize new compounds with the potential to inhibit the growth of these microorganisms. In this sense, a class of molecules called chalcones has shown great versatility for demonstrating broad biological properties, including antifungal activity among them. Through a virtual screening methodology, a chalcone derivative named compound 3 was identified by our group as a promising inhibitor of Paracoccidioides spp. Thus, in order to understand the mode of action of compound 3, we applied a proteomic approach to identify the induced and repressed proteins of P. brasiliensis in the presence of compound 3. In addition, validation assays were performed on the results found. The analysis indicated that the compound can cause an imbalance in the fungal energy homeostasis by reducing the activity of the glycolytic pathway, beta-oxidation and citric acid cycle. In vitro validations also demonstrated that reactive oxygen species accumulate within the cells during exposure to compound 3, which can destabilize cellular components such as the plasma membrane. The molecular docking assay between compound 3 and the enzyme dihydropteroate synthase, which had its expression induced after the treatment, suggest that compound 3 may act as an inhibitor of this protein, impairing the folate biosynthesis that participates as a cofactor in synthesis of nucleotides and some amino acids. Therefore, these data support the efficiency of compound 3 in producing imbalances in key pathways for the P. brasiliensis' metabolic maintenance, contributing to its antifungal role.Item Caracterização da produção científica sobre DNA barcoding no estudo de plantas(Universidade Federal de Goiás, 2021-04-22) Batista, Eliane Cotrim; Gonçalves, Ariany Rosa; http://lattes.cnpq.br/6401185899301616; Telles, Mariana Pires de Campos; http://lattes.cnpq.br/4648436798023532 ; Telles, Mariana Pires de Campos; Nunes, Rhewter; Braga, Ramila dos SantosThe application of the DNA barcoding has been shown to be successful in studies of biodiversity surveys, biomonitoring of invasive species, forensic analysis to assess food security and medicinal products based on medicinal plants, monitoring the illegal trade in threatened species, studies about food ecology, conservation initiatives, etc. The present study had as main objective to raise and systematize the scientific production on the use of DNA barcoding in plants. For this purpose, a search was carried out in the Web of Science database, using as keywords combined the regions annotated in the chloroplast and nuclear genome of Arabidopsis thaliana (model species), which are used as DNA barcoding markers, associated with the expressions: “DNA barcoding” OR “barcoding” OR “DNA barcode” OR “barcode” OR “metabarcoding”. The search was carried out covering the complete period of publication of the database until 2020. The recovered files were filtered, being considered only original articles, excluding review articles, as well as those that did not correspond to the scope of the study. A total of 1,897 articles were retrieved, of which 1,073 were within the criteria’s inclusion. As the first article was published in 2005, the analysis covered the period between 2005 and 2020. The studies were published in a total of 309 sources, with a general average of publication per year equal to 5.21. Scientific production varied over the period, but always with an increasing trend. The average number of authors per document was 3.49, since each article mostly has more than one author, which explains the average number of coauthors per document (5.34). However, out of a total of 3,747 registered authors, the number of articles per author was low, with an average equal to 0.29. China stands out as the main country, according to the origin of the authors, who has contributed with works involving the application of the DNA barcoding technique in the study of plants, being also the most cited country. The most relevant authors, based on the number of publications, were the Chinese: Chen SL, Song J, Yao H, with the article by Chen et al. (2010) the second most cited globally. The journals that showed greater prominence in relation to the number of articles published, considering the subject, were respectively: Plos One, Molecular Ecology Resources and Scientific Reports. It was possible to characterize four groups of documents regarding the use / application of the technique, respectively: Description/delimitation of species (545), Forensic Genetics (275), Development/methodology (163), Ecology (90) and 75 articles were categorized in up to two different groups. The studies grouped in the most prominent category (Description/delimitation of species), range from delimiting species from complex groups to the validation of species with properties of economic interest and species already cataloged in herbariums. The application possibilities have been expanded due to the development of High Throughput Sequencing technologies, opening doors for studies applied to Ecology, Evolution and Forensic Genetics and which use in addition to DNA barcoding, the metabarcoding.Item Citogenômica de pterodon pubescens e citogenética comparativa com P. emarginatus (leguminosae)(Universidade Federal de Goiás, 2020-03-05) Albernaz, Victória Borges; Souza, Luiz Gustavo Rodrigues; http://lattes.cnpq.br/3626102935973855; Soares, Thannya Nascimento; http://lattes.cnpq.br/5590256762396056; Soares, Thannya Nascimento; Harand, Andrea Pedrosa; Vianello, Rosana PereiraThe genus Pterodon Vogel (Leguminosae) has only four species, of which P. pubescens (Benth.) Benth. and P. emarginatus Vogel (both known as "white sucupira") are the closest phylogenetically. These species have a wide geographical distribution in Brazil and have chromosome number 2n = 16 with small and morphologically similar chromosomes. The objective of the present work was to carry out a comparative analysis of the genome size, the banding pattern and the composition of repetitive elements in the chromosomes of P. pubescens and P. emarginatus, aiming to enhance the cytogenomic and evolutionary knowledge of these species. For this, cytogenetic characterization was performed by analyzing the number and chromosomal morphology, CMA and DAPI banding, hybridization with DNAr 5S and 35S and determining the genome size by flow cytometry. In addition, we used P. pubescens genome sequencing by NGS (Next Generation Sequencing) using the Illumina platform to characterize repetitive genomic fractions, using a Galaxy/RepeatExplorer-Elixir platform. The most abundant elements of the P. pubescens genome were located on the chromosomes by fluorescent in situ hybridization (FISH) and transferred to P. emarginatus. The species showed very similar karyotypes with: (i) CMA+/DAPI- bands in the terminal region of two chromosome pairs and pericentromeric region of all chromosomes; (ii) two pairs of 35S rDNA sites co-located with the terminal CMA+ bands; (iii) a pair of 5S rDNA sites located in the proximal region of a chromosomal pair. The genome size of P. pubescens and P. emarginatus was also similar, 1C = 0.665 pg and 1C = 0.620 pg, respectively. The repetitive fraction represented 26,4% of the P. pubescens sequenced genome, with Ty3-Athila Ty3-Athila (24,24%), Ty3-Tekay (21,93%) and Ty1-Ale (3,37%) retrotransposons being the most abundant elements. Low abundance satellite DNAs were identified: PubSat1-254 (2,09%), PubSat2-76 (2,06%), PubSat3-216 (0,58%), PubSat4-138 (0,23%). In situ hybridization reveal that all analyzed repeats were enriched in proximal CMA+ heterochromatin in both species, except for the Ty1-Ale retroelement, which was also dispersed also in the euchromatin of P. pubescens chromosomes. The cytomolecular similarity observed here suggests that the genomes of P. pubescens and P. emarginatus have highly similar repetitive fractions, which corroborates their phylogenetic proximity. However, the recent expansion of the Ty1-Ale element in the P. pubescens genome suggests some degree of differentiation in the repetitive fractions of these genomes.Item Identificação e caracterização de microRNAs no fungo dimórfico Histoplasma capsulatum(Universidade Federal de Goiás, 2020-03-06) Fernandes, Lucas Barros; Curcio, Juliana Santana de; http://lattes.cnpq.br/9577753232871923; Soares, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637; Tomazett, Mariana Vieira; Pereira, Maristela; Soares, Célia Maria de AlmeidaMicroRNAs are small non-coding RNA molecules involved in the RNA interference phenomenon. These small RNAs contain between 18 and 25 nucleotides and are derived from precursors containing a hairpin structure. Their biogenesis and mode of action are well understood in plants and animals, but in fungi their biogenesis is still poorly understood. However, small RNAs similar to microRNAs (miRNAs-like) have been identified in various fungi, such as Neurospora crassa, Sclerotinia sclerotiorum, Penicillium marneffei, Trichoderma reesei and Paracoccidioides brasiliensis. Even though the amount of data on these molecules in the Fungi kingdom has been increasing, up to now there are no descriptions of microRNAs in important human pathogens such as Histoplasma capsulatum. Therefore, due to the absence of the characterization of this regulatory mechanism in this human pathogen, this study aimed to identify microRNAs in this pathogen and to infer the biological processes regulated by these interference RNAs in this fungus. In order to do so, the methodology employed in this study was the search for published microRNAs sequences for other fungi in the literature and in silico analysis to predict the homology of these known sequences to genome regions of two H. capsulaum strains H88 and G217B. After the bioinformatics analysis, we identified six microRNAs in H. capsulatum H88 and four microRNAs in H. capsulatum G217B .One of the microRNAs was conserved in both strains. This microRNA was selected for target investigation. We identified 551 targets in the H88 strain and 495 targets in the G217B strain. Biological processes identified as being regulated by this microRNA comprise the synthesis and the degradation of cell wall components, the energetic metabolism and DNA processing. The results imply the presence of microRNAs in this fungi and a high number of targets for each microRNA, which hints on the existence of a complex network of microRNA post-transcriptional regulation in H. capsulatum.Item Caracterização do antígeno proteico SsaA de Staphylococcus saprophyticus utilizando estratégias in silico e modelo ex vivo de infecção(Universidade Federal de Goiás, 2020-03-02) Silva, Guilherme Algusto Alves; Rocha, Juliana Alves Parente; http://lattes.cnpq.br/7089231795367245; Rocha, Juliana Alves Parente; Tomazett, Mariana Vieira; Amaral, André CorrêaStaphylococcus saprophyticus is a Gram-positive bacterium and stands out as the second pathogen responsible for diagnosed cases of urinary tract infection (UTI), affecting mainly young women. Some factors may explain the ability of S. saprophyticus to colonize periurethral, urinary and genital regions, such as the ability to bind to the epithelial tissue of the genitourinary tract and the high activity of the urease enzyme. However, few mechanisms that this bacterium uses to efficiently infect and colonize the host are fully elucidated. In species of the Staphylococcus genus, most of the known virulence factors are proteins or the pathogen's surface. Our research group identified proteins secreted from S. saprophyticus isolates that demonstrated a strong ability to stimulate the immune response in mice and one of the main immunogenic proteins identified was the Staphylococcal Antigen Secreted A (SsaA), not yet characterized in this species. In other species of the Staphylococcus genus, the SsaA protein seems to be related to virulence factors regulated by the same systems, but its specific role during infection has not yet been fully elucidated. In this sense, we propose the characterization of the SsaA protein in S. saprophyticus. Bioinformatics analyzes using the database revealed that the SsaA protein has a CHAP domain with an expected amidase function. The 3D structure of the SsaA protein was predicted through the modeling of proteins using an online server following the validation protocols, the ABCpred server was used to predict epitopes in the SsaA protein. Analysis of the phagocytosis assay revealed that blocking the SsaA protein by sera containing anti-SsaA antibody reduces the number of cells recovered thus indicating that SsaA may be important during the infectious process. Our work will contribute to elucidate the biological function of the SsaA protein, an immunogenic protein that can be useful as a diagnostic target and / or vaccine in this model.Item Regra das Ilhas e genética quantitativa evolutiva do tamanho corporal em Elephas maximus borneensis(Universidade Federal de Goiás, 2020-03-24) Silva, Felipe Naves; Diniz Filho, José Alexandre Felizola; http://lattes.cnpq.br/0706396442417351; Diniz Filho, José Alexandre Felizola; Telles, Mariana Pires de Campos; Jardim, Lucas Lacerda Caldas ZaniniThe Islands Rule describes that there is a tendency, especially in mammals, that species that are large on the continent tend to suffer from dwarfism when isolated on an island, while those that have small size on the continent tend to suffer from gigantism. Elephas maximus borneensis is small in size compared to other elephants of the genus, and there is also controversy about its condition as a natural subspecies of Borneo, when it possibly arrived on the island and the degree of isolation. For this reason, starting from the Evolutionary Quantitative Genetic Model based on the individual, we incorporate a more realistic parameterization, assuming the dioecious subspecies with sexual reproduction, balanced sexual proportion and random monogamous mating, with generations without overlapping. In addition, we use the adaptive peak on the pre-established island and also evolutionary characteristics for island populations such as: heritability, migration, inbreeding and mutation. We simulated four scenarios based on the hypotheses of colonization and evolution of the elephant's body size, taking into account the time of colonization and the possible ancestral body weight. We compared the results of the simulations with the molecular data of this subspecies and the historical records. We conclude then that among the four scenarios, only the third was supported by all the data assumed in the present study, thus, the most accepted hypothesis, describing that possibly the subspecies of Elephas maximus borneensis suffered an isolation of 11 thousand to 18 thousand years in Java and, about 700 years ago, was subjected to two subsequent founding events, arriving then on the Island of Borneo.Item Estrutura genética espacial intrapopulacional em Stryphnodendron adstringens (barbatimão fabaceae)(Universidade Federal de Goiás, 2020-04-13) Miranda, Alline Afonso do Nascimento Creado de; Telles, Mariana Pires de Campos; ttp://lattes.cnpq.br/4648436798023532; Telles, Mariana Pires de Campos; Braga, Ramilla dos Santos; Pinto, Rafael BarbosaThe Cerrado biome, despite containing one of the greatest biodiversity on the planet, undergoes constant deforestation due to the expansion of agricultural frontiers. The loss of natural territory consequently leads to the loss of genetic diversity. This is worrying when it comes to native species, such as Stryphnodendron adstringens, popularly known as barbatimão. Therefore, it is important to know the characteristics of the biology of S. adstringens, in order to define efficient management and conservation strategies. In this context, the objective of this study was to evaluate genetic diversity, genetic flow (estimating the size of the neighborhood group), the presence of clones and the spatial genetic structure existing in a natural population of S. adstringens. For this, genomic DNA was extracted from the leaf tissue of 124 individuals, all georeferenced. For the amplification of the microsatellite regions, nine pairs of primers developed for the species were used. Based on the genotype matrix, analyzes of genetic diversity, effective size of inbreeding, cross-fertilization rate, spatial genetic structure and identity analysis were performed to verify the occurrence of clones. For the nine loci, a total of 74 alleles were found and an average of 8.2 alleles per location. The observed average heterozygosity (HO) was equal to 0.556 and the average genetic diversity (HE) was equal to 0.624. The inbreeding coefficient (f) was significant and equal to 0.110, showing an excess of homozygotes. The genetic diversity in the population was considered high, with 71% of the maximum expected heterozygosity. The crossing system was considered mixed based on the cross fertilization rate (t ̂a) equal to 0.80. The effective size of the consanguinity (Nef) was 111.72; corresponding to a genetic representation of 0.90, indicating that there is no genetic deviation in the population. There was a significant spatial genetic structure in the first distance class with pairs of up to 49 meters. A Sp statistic equal to 0.0095 was considered moderate and associated with the genetic neighborhood (Nb = 105.3) demonstrated that pollen dispersion is efficient to maintain genetic diversity in the population. Ten groups of individuals with identical genotypes were found for nine sites used, suggesting the occurrence of vegetative reproduction. The results plan was possible, which, for the formation of germplasm banks, is collected at a minimum distance of 49 meters between the guaranteed items, in addition to contemplating the genetic diversity present in the population.Item Análise proteômica do fungo patogênico humano Fonsecaea pedrosoi, cultivado nas condições de temperatura de 22º C e 36º C(Universidade Federal de Goiás, 2017-09-11) Lima, Davi Vinícius de; Bailão, Alexandre Melo; http://lattes.cnpq.br/5415221996976886; Bailão, lexandre Melo; Paccez, Juliano Domiraci; Novaes, EvandroThe Polymorphic fungus Fonsecaea pedrosoi is the main etiological agent of Chromoblastomycosis (CBM), characterized as a chronic cutaneous mycotic infection, occurring mainly in tropical regions. The limited availability of effective therapeutic protocols coupled with CBM clinical polymorphism lead to often prolonged and high relapse rate therapies. Results regarding the molecular biology of F. pedrosoi are scarce however, some virulence factors have already been described, evidencing the need to explore the mechanisms used by this pathogen during infection. The present work carried out in silico analyzes under specific culture conditions, with temperatures of 22º C and 36º C, aiming to better understand the proteome of this fungus, both intracellular and secreted. In order to identify these proteins, we used bioinformatic tools that are available online and allowed the prediction of protein sets that can be secreted by classical and non-classical routes. After the in silico prediction method, proteins were identified in the fungal extracts by the Ultraperformance Liquid Chromatography coupled to Mass Spectrometry (UPLC-MSE) methodology. Those identified in the extracts that brought a more reliable interpretation corroborated the results in silico. The proteins identified stand out for being related to various biological processes, such as metabolism of carbon compounds, energy, proteins related to thermal and oxidative stress, defense, proteins related to the cell cycle, among other functions. The analyzes showed that the vast majority of identified proteins are secreted by alternative pathways, and the results point to the great importance of the biological roles that these secreted proteins can play during the onset of infection. Thus, we observed that the fungus presents variations in protein expression levels according to the condition addressed and may reflect on its behavior and responses to the successful establishment of the infection.Item Métodos diagnósticos para o vírus mayaro: revisão sistemática e avaliação molecular em pacientes arbovirose like em unidade municipal de sáude de Goiânia-Goiás(Universidade Federal de Goiás, 2020-02-17) Abrantes, Gabrielly Regis; Anunciaçāo, Carlos Eduardo; http://lattes.cnpq.br/4354412874919580; Lacerda, Elisângela de Paula Silveira; http://lattes.cnpq.br/9390789693192751; Lacerda, Elisângela de Paula Silveira; Anunciaçāo, Carlos Eduardo; Lino, Guilherme; Mrué, FátimaDiseases caused by viruses are considered an often neglected public health problem that affects thousands of people worldwide, every year. Symptoms like fever, arthralgia and rash. classify patients as syndromic dengue or arbovirus like. The differential diagnosis of arbovoresis is by serological and molecular techniques. The present study aimed to evaluate diagnostic techniques in studies published in the literature in order to develop a Systematic Literature Review on the detection of the Mayaro virus that allows health professionals to carry out an efficient laboratory diagnosis, as well as the application of molecular tests for the diagnosis of MAYV. The available literature was evaluated by systematic review, focusing on the diagnosis of the Mayaro virus in humans and its methodologies. The systematic review was carried out through an exploratory study in the databases: Scielo (Scientific Eletronic Library Online), Medline (Medical Literature Analysis and Retrieval System Online), Scopus database, PubMed, LILACS from June 8, 2019 to 02 November 2019, selecting scientific articles according to the criteria of the PRISMA (2009) recommendation. The data showed that the circulation of the Mayaro virus in urban regions is already well established worldwide and that the misdiagnosis is frequent due to similar symptoms with other arboviruses. There is a wide use of varied methods over time in the diagnosis for this arbovirus, requiring further research to implement a standardized, sensitive and specific method. In a second stage of the research, between May to August 2017 and January to June 2018, 452 samples of patients with syndromic dengue symptoms (arboviruses like) were screened. The samples were sent to the Molecular Genetics and Cytogenetics Laboratory of the Federal University of Goiás, where they were subjected to molecular screening by Polymerase Chain Reaction followed by Reverse Transcriptase (RT-PCR) reaction. All positive cases for the first PCR were confirmed on RT-qPCR for viral detection. Thirteen positive samples went through both tests and thus were subjected to Sanger sequencing. In this experimental study, 17 samples were positive for the Mayaro virus infection (RT-PCR and RT-qPCR), and co-infections with the Dengue virus were also found by molecular screening carried out in parallel for the Dengue virus by RT-PCR and RT-qPCR. The positive samples for MAYV / DENV were analyzed phylogenetically, suggesting circulation of Dengue serotype 2 and a Peruvian strain of Mayaro Virus (strain IQ 4235) in Goiânia.Item Alterações metabólicas resultantes da fosforilação de Isocitrato Liase em Paracoccidioides lutzii(Universidade Federal de Goiás, 2015-04-08) Silva, Karla Christina Sousa; Tauhata, Sinji Borges Ferreira; http://lattes.cnpq.br/7082495215620618; Tauhata, Sinji Borges Ferreira; Bailão, Alexandre de Melo; Brito, Wesley de Almeida; Souza, Guilherme Rocha Lino de; Faria, Fabrícia Paula deParacoccidioidomycosis (PCM) is a systemic mycosis endemic from Latin America caused by the species encompassed in Paracoccidioides genus. This mycosis affects mainly economically active men, who live in rural areas. PCM may cause severe wounds to individuals and is highly incapacitating. This disease is considered a neglected disease by World Health Organization (WHO). When in a hostile environment, like deprivation of carbon sources (6C), fungi tend to use alternative strategies to assist the energetic metabolism. One strategy is using the glyoxylate cycle, which consists in 5 steps, 3 of them being common to the tricarboxylic acid cycle and the other 2 steps are catalyzed by exclusive enzymes from the glyoxylate cycle: isocitrate lyase (ICL) and malate synthase. There are reports in the literature suggesting that phosphorylation of ICL ofis a possible mechanism of enzyme regulation. Phosphorylation is a post-translational ubiquitous ubiquitous and known to modulate the activity / action of many molecules of diverse organisms. In this work, through the use of molecular interactions assays (pull down) using ICL of Paracoccidioides lutzii in its phosphorylated state and non-phosphorylated We found evidence, which suggest that the purine biosynthesis pathway is being used as an alternative way to produce energy to the organism and a high involvement of ICL with amino acids biosynthesis on its non-phosphorylated state. Apparently, phosphorylation works marking ICL to become degraded. This observation may open a new research field through the elaboration of new drugs for PCM treatment using phosphatase inhibitors.Item Avaliação molecular dos polimorfismos genéticos na susceptibilidade à nefropatia diabética por alterações hemodinâmicas e endoteliais(Universidade Federal de Goiás, 2018-12-19) Anjos, Laura Raniere Borges dos; Santos, Rodrigo da Silva; http://lattes.cnpq.br/4806187026900959; Reis, Angela Adamski da Silva; http://lattes.cnpq.br/3243656364470085; Cruz, Aparecido Divino da; Cruz, Aline Helena da Silva; Rocha, Thiago Lopes; Ternes, Yves Mauro FernandesDiabetic nephropathy (DN) is the major microvascular complication of Diabetes mellitus (DM) and is among the leading causes of kidney disease. This pathology is important because of glomerular changes induced by imbalance of glucose homeostasis and high intraglomerular pressure. Studies indicate that diseases are influenced not only by the environment but also by genetic factors. Among the genetic determinants, the polymorphisms in the MTHFR and VEGF genes stand out. These genes are oriented towards the synthesis of the enzyme methylenetetrahydrofolate reductase (MTHFR) and vascular endothelial growth factor (VEGF), respectively. The MTHFR enzyme plays an important role in the processing and processing cycle of methionine. VEGF is an important protein involved in signaling that stimulates vasculogenesis and angiogenesis. Thus, the objective of this study was to perform a molecular evaluation of MTHFR C677T and VEGF -141 A → C single nucleotide polymorphisms (SNPs) in diabetic patients with microvascular complications caused by hemodynamic and endothelial changes in a Brazilian population. A total of 345 were genotyped for polymorphisms using PCR-RFLP. The advanced RStúdio environment was used for statistical analysis. The logistic regression of the gene polymorphism in the MTHFR gene increases the risk (OR = 2.57, p = 0.003) of the diabetic individual developing ND. The polymorphism in VEGF gene reveals a 3.74-fold risk for DN (p = 0.001) in the eventual condition of diabetic patient. When analyzing the association of clinical variables and polymorphism in the MTHFR gene, the results showed no correlation. The association of clinical variables with the VEGF gene polymorphism, the results indicated the same in creatinine, in GFR and in diastolic blood pressure. Clinical associations observe a mechanism underlying the role of polymorphisms in renal dysfunction. Results suggest that polymorphisms MTHFR gene confer susceptibility to ND in the patients with DM.Item Análise da herança do DNA do Trypanosoma cruzi em parentais e progênies de chagásicos de famílias de São Felipe – Bahia(Universidade Federal de Goiás, 2019-02-26) Santos, Eduardo José dos; Cardoso, Clever Gomes; http://lattes.cnpq.br/9545455455623006; Lacerda, Elisângela de Paula Silveira; http://lattes.cnpq.br/9390789693192751; Teixeira, Antônio Raimundo Lima Cruz; Zapata, Marco Túlio Antônio GarciaThe Trypanosoma cruzi, etiologic agent of Chagas disease is a flagellate protozoan transmitted by insect bugs (Reduvidae:Triatomine) to humans and wild mammals. The T. cruzi contains DNA in the nucleus (nDNA) and in the mitochondrion (kDNA). The kinetoplastid protozoan comprises maxicircles and and minicircles kDNA network sequences that integrate in the host’s cell genome. Insertional integrations of kDNA sequences bear high affinity to retrotransposable LINE-1 randomly distributed in the genome of somatic and germ line cells, and, thus generate an increasing diversity due to recombination and reshuffling. However, the profiles of kDNA integrations into the human genome in different Brazilian Ecosystems are unknown. In this regard, the ratio of transfer of T. cruzi minicircles sequences into the human genome was south. The study consisted of analyses of 36 members of two families from São Felipe-Bahia where Chagas disease is endemic. The study showed that 22% (8/36) families’ members had the diagnoses of the disease by immunofluorescence (IF) and enzyme-linked immunosorbant assay (ELISA). Yet, the PCR assays showed 61% (22/36) of families’ members had kDNA and nDNA assays positive for the T. cruzi infection. In order to detect the kDNA integration sites in the human genome, a tpTAIL-PCR (Targeted Primer Thermal Assymetric Interlaced-PCR), which consisted in combination of primers derived from T. cruzi minicircle conserved sequence with human LINE-1 sequence derived primers, was employed. The amplicon sequences obtained were subjected to homology analyses in Blast software. The hybrid kDNA-human DNA sequences revealed lateral and vertical transfer of T. cruzi minicircles into the genome of Chagas parents and progeny. Unravel of dynamics of mutations contributes to define variable profiles of kDNA minicircle sequence insertions into the human genome at different Ecosystems.Item Diversidade genética de Eugenia dysenterica e sua correlação com a riqueza de Eugenia (Myrtaceae) no cerrado(Universidade Federal de Goiás, 2016-03-04) Oliveira, Hauanny Rodrigues; Staggemeier, Vanessa Graziele; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4759933P0; Diniz Filho, José Alexandre Felizola; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4727587J2; Diniz Filho, José Alexandre Felizola; Rangel, Thiago Fernandes Lopes Valle de Brito; Nabout, João CarlosBrazilian Cerrado is a biodiversity hotspot due to high levels of species diversity and endemism. However, in the last decades its biodiversity has been negatively affected by human actions, which can also be matched with climate changes. To assess the processes raising and holding biodiversity are important as bases to elaborate conservationist tools. Thus, our aims were (i) to describe the richness of Eugenia in Cerrado, and (ii) to correlate genetic diversity of Eugenia dysenterica and species diversity of Eugenia, to answer if diversity at the two hierarchical levels responds to the ecological and evolutionary process in the same way. Myrtaceae is the eighth largest plant family in the world and it is a good model for studies in biodiversity because a detailed knowledge has been accumulated about its distribution, taxonomy and phylogeny. Moreover, Eugenia is one of the most representative genera in Myrtaceae, its species has social, economic and ecological importance. We found that richness pattern of Eugenia changed over time with an evident displacement from the southwest in the past times to southeast of Cerrado nowadays (Chapter 1). We also predicted that 93% of Eugenia species in the Cerrado will lose range size in different scenarios for the future. We found that diversity levels are responding in different ways to the process (Chapter 2), mainly due to climate shifts during the Last Glacial Maximum. Furthermore, clades more phylogenetically distant of E. dysenterica were negatively correlated with its genetic diversity, hence species with recent histories exhibit higher ecological and evolutionary differences reflecting in divergent responses compared with early-branched clades. The positive correlation found between species diversity of clade two and genetic diversity of E. dysenterica, revealed that closely related species respond to the eco and evolutionary process in the same way.