53 resultados para análise genético-molecular


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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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The northeastern region is responsible to 14.32% of sugarcane national production. This lowered contribution is due to edaphoclimatic condition. Flowering is a vital process to plant which consumes lots of energy and it culminates in a process called isoporization. This one can give in a decreasing of 60% on alcohol and water production. It may consider that cropped sugarcane has a hibrid with octaploid genome, there are varieties with a flowering standard until of non flowering. Using this natural genetic potential on different croppings of sugarcane, the aim of this work was to understand as this process occurs by the usage of subtractive approaches. The total RNA was extracted using Trizol of peaks of merisematics of croppings with induced flowering and other with late flowering. From this total RNA were built four subtractives libraries (B1- induced early flowering subtracted on late flowering not induced; B2- late flowering not induced subtracted induced early flowering; B3- induced early flowering subtracted of not induced early flowering; B02- not induced early flowering subtracted from induced early flowering) using kits Super Smart cDNA synthesis and BD Clontech kit select cDNA subtraction (Clontech). This material was clone don vector pGEM T-easy(Promega) and changed in competent cells of E.coli DH10B. Given analysis sequence was carried out a program BLASTn against database of NCBI and genome of Arabidopsis thaliana, rice and maize. Clones were grouped in 9 different classes according to function. Some factors already related as couples of flower induction were identified at different libraries. And grouped proteins with cell cycle and it controls were presents, mainly kinases proteins. Related factors to proteic sinthesis, metabolism, defence, cell communication were also given in both libraries .Some identified genes did not show similarity on database or homology with hypothesis function, and it can represents new genes to be deposited in international database. These results offers that some identified on sugarcane, classified as on factors classes, cell cycle and cell communication, trough unknown genes, can be linked with genetic changing to the flowering process found in the northeastern region

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

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Pleomorphic adenoma and adenoid cystic carcinoma represent a benign and malignant salivary gland neoplasm, respectively, that shares the same histological origin, however with distinct biological behavior. The aim of the present study was identify the -160 C/A polymorphism in the gene CDH1, mutational analysis of CTNNB1 gene and evaluation the expression of the E-cadherin and β-catenin in pleomorphic adenomas and adenoid cystic carcinomas. Furthermore, it was proposed correlate the immunochemistry staining patterns with the polymorphism and mutations. Twenty-four pleomorphic adenomas and 24 adenoid cystic carcinomas were retrieved. The polymorphism analysis was performed by restriction fragment length polymorphism (RFLP), using the restriction enzymes HphI or AflIII and the mutational screening was performed by PCR-single strand conformational polymorphism (PCR-SSCP). The immunohistochemical analysis was taken by the counting of cells, recorded as the Hscore index, and considering the presence or absence, intensity, distribution and localization of proteins expression. Comparing the two neoplasms, the results demonstrated statistically significant difference for the E-cadherin and β-catenin expression, with pleomorphic adenoma presenting weaker immunostaining. Was observed statistical correlation between E-cadherin and β-catenin expression. CDH1 heterozigotic polymorphism was seen in two cases and 13 cases displayed abnormal mobility electrophoretic shifts, suggesting CTNNB1 gene mutation. The immunohistochemical expression was not statistically correlated with the polymorphism or suggested mutations. In conclusion this study supports that the E-cadherin/β-catenin complex immunohistochemical expression might be related with the myoepithelial component amount and differentiation neither the tumor biological behavior. The cases that showed E-cadherin gene polymorphism presented reduced protein expression and, moreover, CTNNB1 suggested mutations seem not influence in the β-catenin protein expression

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The main specie of marine shrimp raised at Brazil and in the world is Litopenaeus vannamei, which had arrived in Brazil in the `80s. However, the entry of infectious myonecrosis virus (IMNV), causing the infectious myonecrosis disease in marine shrimps, brought economic losses to the national shrimp farming, with up to 70% of mortality in the shrimp production. In this way, the objective was to evaluate the survival of shrimps Litopenaeus vannamei infected with IMNV using the non parametric estimator of Kaplan-Meier and a model of frailty for grouped data. It were conducted three tests of viral challenges lasting 20 days each, at different periods of the year, keeping the parameters of pH, temperature, oxygen and ammonia monitored daily. It was evaluated 60 full-sib families of L. vannamei infected by IMNV in each viral challenge. The confirmation of the infection by IMNV was performed using the technique of PCR in real time through Sybr Green dye. Using the Kaplan-Meier estimator it was possible to detect significant differences (p <0.0001) between the survival curves of families and tanks and also in the joint analysis between viral challenges. It were estimated in each challenge, genetic parameters such as genetic value of family, it`s respective rate risk (frailty), and heritability in the logarithmic scale through the frailty model for grouped data. The heritability estimates were respectively 0.59; 0.36; and 0.59 in the viral challenges 1; 2; and 3, and it was also possible to identify families that have lower and higher rates of risk for the disease. These results can be used for selecting families more resistant to the IMNV infection and to include characteristic of disease resistance in L. vannamei into the genetic improvement programs

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Among the traits of economic importance to dairy cattle livestock those related to sexual precocity and longevity of the herd are essential to the success of the activity, because the stayability time of a cow in a herd is determined by their productive and reproductive lives. In Brazil, there are few studies about the reproductive efficiency of Swiss-Brown cows and no study was found using the methodology of survival analysis applied to this breed. Thus, in the first chapter of this study, the age at first calving from Swiss-Brown heifers was analyzed as the time until the event by the nonparametric method of Kaplan-Meier and the gamma shared frailty model, under the survival analysis methodology. Survival and hazard rate curves associated with this event were estimated and identified the influence of covariates on such time. The mean and median times at the first calving were 987.77 and 1,003 days, respectively, and significant covariates by the Log-Rank test, through Kaplan-Meier analysis, were birth season, calving year, sire (cow s father) and calving season. In the analysis by frailty model, the breeding values and the frailties of the sires (fathers) for the calving were predicted modeling the risk function of each cow as a function of the birth season as fixed covariate and sire as random covariate. The frailty followed the gamma distribution. Sires with high and positive breeding values possess high frailties, what means shorter survival time of their daughters to the event, i.e., reduction in the age at first calving of them. The second chapter aimed to evaluate the longevity of dairy cows using the nonparametric Kaplan-Meier and the Cox and Weibull proportional hazards models. It were simulated 10,000 records of the longevity trait from Brown-Swiss cows involving their respective times until the occurrence of five consecutive calvings (event), considered here as typical of a long-lived cow. The covariates considered in the database were age at first calving, herd and sire (cow s father). All covariates had influence on the longevity of cows by Log-Rank and Wilcoxon tests. The mean and median times to the occurrence of the event were 2,436.285 and 2,437 days, respectively. Sires that have higher breeding values also have a greater risk of that their daughters reach the five consecutive calvings until 84 months

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The catalytic cracking of triglycerides presents itself as a possible alternative to the production of biofuels with low emission of pollutants. In this work were synthesized the SAPO-5, the catalysts for the cracking reaction of soybean oil is presented. The solids were powder X-ray diffraction (XRD), thermogravimetric analysis (TG/DTG) and infrared spectroscopy (FTIR). The analyses indicated that the synthesis method has employed to obtain materials with high surface area and high acid. The soybean oil thermal and thermal catalytic cracking, realized from the room temperature to 450 ºC in a simple distillation system, has allowed obtaining two liquid fractions, each consisting of two phases, one aqueous and another organic, organic liquid (OL). The OL obtained from first fractions has shown high acid index, even in the thermal catalytic process. The products obtained in the cracking of soybean oil were analyzed by distillation, acid number, infra-red spectroscopy, density, viscosity, carbon residue, cetane number determination and characterization. The analysis of the products obtained in the presence and in the absence of the SAPO-5 permitted to conclude that all the solids tested presented catalytic activity in the deoxygenation of final products only at the second step of the cracking process

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Dispersions composed of polyelectrolyte complexes based on chitosan and poly(methacrylic acid), PMAA, were obtained by the dropping method and template polymerization. The effect of molecular weight of PMAA and ionic strength on the formation of chitosan/poly(methacrylic acid), CS/PMAA, complexes was evaluated using the dropping method. The increase in molecular weight of PMAA inhibited the formation of insoluble complexes, while the increase in ionic strength first favored the formation of the complex followed by inhibiting it at higher concentrations. The polyelectrolyte complexation was strongly dependent on macromolecular dimensions, both in terms of molecular weight and of coil expansion/contraction driven by polyelectrolyte effect. The resultant particles from dropping method and template polymerization were characterized as having regions with different charge densities: chitosan predominating in the core and poly(methacrylic acid) at the surface, the particles being negatively charged, as a consequence. Albumin was adsorbed on templatepolymerized CS/PMAA complexes (after crosslinking with glutardialdehyde) and pH was controlled in order to obtain two conditions: (i) adsorption of positively charged albumin, and (ii) adsorption of albumin at its isoelectric point. Adsorption isotherms and zeta potential measurements showed that albumin adsorption was controlled by hydrogen bonding/van der Waals interactions and that brushlike structures may enhance adsorption of albumin on these particles

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The Amazon holds over half of the planet's remaining tropical forests and comprises the largest biodiversity in the world, accounting for approximately 60 % of the Brazilian territory. However, deforestation fires in the region causes serious problems to exposed human. The aim of this study was to evaluate the chemical compounds as well as the cellular and molecular effects after exposure to organic material extracted from particulate matter less than 10 µm (PM10) in the Amazon region. As for the chemical composition, n-alkanes analysis showed a prevalence of anthropogenic influence during the fires in the region. In addition, there was a predominance of monosaccharides from biomass burning markers. Also, the Polycyclic Aromatic Hydrocarbons (PAH) and their derivatives have also been identified in samples collected in the Amazon. By using the PAH concentrations was possible to calculate the BaP-equivalent and it was found that the dibenz(a) anthracene contributes with 83% to potential carcinogenic risk. As for the potential mutagenic risk, the benzo (a) pyrene is the HPA that has a major contribution in this analysis. It may be noted that the retene was the most abundant PAH. This compound was genotoxic and cause death by necrosis in the human lung cells. In biological tests, the data showed that organic PM10 is capable of causing genetic damage in both plant cells and in human lung cells. This damage cause an arrest in the G1 phase of the cell cycle exposed, increasing the expression of p53 and p21. Additionally, the PM10 caused cell death by apoptosis, increasing the foci of histone - H2AX. Given these results, it is important to emphasize the reduction and better control of biomass burning in the Amazon region thus improving the quality of health of the population being exposed. As clearly stated recently by the World Health Organization, the reduction of air pollution could save millions of lives annually.

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Background & Aims: HFE-associated Hereditary Hemochromatosis (HH) is one of the most frequent autosomal recessive disease in the caucasian population, caused by the high absorption and deposition of iron in several organs. This accumulation results in several clinical complications such as cirrhosis, arthritis, cardiopathies, diabetes, sexual disorders and skin darkening. Although most of the cases are homozygous individuals for the C282Y mutation, another two mutations, H63D and S65C, have been reported to be associated with milder forms of the disease. The objective is to avaluate the distribution of C282Y, H63D and S65C mutations in the HFE gene in patients with suspected HH in the state of Rio Grande do Norte, Brazil. Methods: Samples of peripheral blood were taken from 335 patients originating from Natal-RN, a city in northeastern Brazil with suspected of HH and which were screened for the HFE gene C282Y, H63D and S65C mutations, using molecular genetics assays (Polymerase Chain Reaction- Restriction Fragments Length Polymorphism). The main criterion for including such patients in the study was the increasing of persistent serum ferritin in individuals aged between 18 and 70 or older, both males and females. As to the exclusion criteria, individuals holding hemolytical anemia, talassemy and previously report of blood transfusion did not take part of the study. Results: Out of the 335 patients studied, 143 patients showed absence of mutation and 195 showed some kind of mutation in the HFE gene: 07/335 (2,08%) were homozigous C282Y, 25/335 heterozygous C282Y, 25/335 (7,46%) were homozigous H63D, 115/335 (34,32%) heterozygous H63D, 5/335 (1,48%) heterozygous S65D, 11/ 335 (3,28%) and were double heterozygous (H63D/C282Y). None patients were Homozygous S65D and S65D heterozygous (S65D/H63D and S65D/C282Y). Conclusions. The distribution of the HFE gene C282Y, H63D and S65C mutations found in our group matches the tendencies observed in other European countries. Due to the high prevalence of hemochromatosis, its seriousness and easy treatment, the genetic diagnosis of HH has become a dream, especially in the high risk group.

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The plant metabolism consists of a complex network of physical and chemical events resulting in photosynthesis, respiration, synthesis and degradation of organic compounds. This is only possible due to the different kinds of responses to many environmental variations that a plant could be subject through evolution, leading also to conquering new surroundings. The glyoxylate cycle is a metabolic pathway found in glyoxysomes plant, which has unique role in the seedling establishment. Considered as a variation of the citric acid cycle, it uses an acetyl coenzyme A molecule, derived from lipids beta-oxidation to synthesize compounds which are used in carbohydrate synthesis. The Malate synthase (MLS) and Isocitrate lyase (ICL) enzyme of this cycle are unique and essential in regulating the biosynthesis of carbohydrates. Because of the absence of decarboxylation steps as rate-limiting steps, detailed studies of molecular phylogeny and evolution of these proteins enables the elucidation of the effects of this route presence in the evolutionary processes involved in their distribution across the genome from different plant species. Therefore, the aim of this study was to establish a relationship between the molecular evolution of the characteristics of enzymes from the glyoxylate cycle (isocitrate lyase and malate synthase) and their molecular phylogeny, among green plants (Viridiplantae). For this, amino acid and nucleotide sequences were used, from online repositories as UniProt and Genbank. Sequences were aligned and then subjected to an analysis of the best-fit substitution models. The phylogeny was rebuilt by distance methods (neighbor-joining) and discrete methods (maximum likelihood, maximum parsimony and Bayesian analysis). The identification of structural patterns in the evolution of the enzymes was made through homology modeling and structure prediction from protein sequences. Based on comparative analyzes of in silico models and from the results of phylogenetic inferences, both enzymes show significant structure conservation and their topologies in agreement with two processes of selection and specialization of the genes. Thus, confirming the relevance of new studies to elucidate the plant metabolism from an evolutionary perspective

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The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.

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Dengue is an acute infectious disease, usually transmitted by Aedes aegypti mosquitoes. The etiologic agents belong to the family Flaviviridae, genus Flavivirus, and occur as four antigenically related but distinct serotypes designated DENV-1, 2, 3, and 4. In Brazil, the disease represents a national public health problem. The purpose of the present study was to describe epidemiological aspects of dengue in the State of Rio Grande do Norte, from 2013 to 2014. A total of 483 blood or serum samples, collected from January 2013 to December 2014, were studied by RT-PCR for viral detection and typing. The infection was confirmed in 36.44% (176/483) of the cases studied. This study detected the circulation of three serotypes of dengue virus in Rio Grande do Norte, DENV-1, 2, and 4. The predominant serotype in 2013-2014 was the DENV-4, representing 83.51% (81/97) and 68.35% (54/79) of the positive cases, respectively. Regarding the spatial distribution, most of the cases occurred in Natal and Caicó, with 9.28% (9/97) and 18.99% (15/79), respectively. The months with the biggest viral circulation in RN were March 2013 and May 2014. The female gender was the most affected, representing 69.07% (67/97) in 2013 and 54.43% (43/79) in 2014. The most affected age groups were 21-30 years (2013) and 11-20 years (2014) with 25.77% (25/97) and 20.25% (16/79) positive cases, respectively. Phylogenetic analysis indicated that genotype V (DENV-1) and genotype II (DENV-4) circulated in the State. Our results provide information about the dynamics of DENV in the Rio Grande do Norte, important for the development of disease control strategies.

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Estresses ambientais abióticos são fatores que causam respostas ao nível molecular, fisiológico e morfológico em plantas, dependendo também de sua intensidade e duração. É visto que algumas espécies apresentam tolerância a condições estressantes e ao mesmo tempo são fontes naturais de matéria prima para indústria. Nesse contexto encontra-se a mamona (Ricinus comunnis L.), principal fonte de óleo de rícino valorizado por suas aplicações farmacêuticas e principalmente industriais, vem sendo usada como cultura em regiões onde a disponibilidade de água é reduzida, usada como fonte de renda para agricultura da região nordeste brasileira. Visto que pouco se sabe sobre as respostas moleculares que levam essa planta a tolerar regiões secas e como as sementes, principais foco de interesse, respondem a essa escassez, nesse trabalho foram construídas duas bibliotecas de cDNAs, onde a partir de uma abordagem subtrativa, continham RNAs diferencialmente expressos em sementes de plantas mamona submetidas ao estresse hídrico durante 5 dias (biblioteca L7), e a outra RNAs diferencialmente expressos em sementes controle (biblioteca L5). A biblioteca L7 apresentou a maior variedade de transcritos com um total de 182. A maior parte das funções estabelecidas pelo sistema Gene Ontology - GO, foram direcionadas aos “Processos Metabólicos” (526), em segundo “Respostas a estímulos” (57), o terceiro termo mais abundante foram referentes a “Desenvolvimento”(26). Já na biblioteca L5, foram encontrados 91 transcritos, com maior parte de suas funções referentes a “Processos Metabólicos”(413), em segundo “Respostas a estímulos” (8) e em terceiro Regulação (6). Alguns dos transcritos da biblioteca L7 foram escolhidos para análise por repetirem-se mais de 3x e não aparecerem na biblioteca L5, o que indica uma possível regulação positiva sobre estresse. As análises sobre Metalotioneína (4x), mostraram que a sequência de proteica apresentava os domínios conservados que a caracterizava como tipo II, onde são encontrados dois domínios funcionais ricos em cisteína com posições altamente conservadas, desempenhando a função de ligar-se a metais pesados, correlacionadas assim como a atividade de eliminação EROs e defesa contra o estresse oxidativo, além de apresentar homologia com a sequência de Bruguiera gymnorhiza, uma planta de mangue adaptada a ambientes salinos. Analisamos também os transcritos da referente a proteína AUXIN-REPRESSED 12.5 KDA (3x), apontada como sendo reprimida pelo hormônio auxina e associada ao processo de dormência da semente, é descrito em uma família gênica onde vários membros pertencem as vias de resposta ao estresse. Por último, analisamos a proteína GLUTELIN TYPE-A 3 (5x), uma importante proteína de armazenamento com caráter hidrofílico, possivelmente direcionada para o vacúolo. Em nosso trabalho foi possível observar um aumento de transcritos em relação a subtração controle, possivelmente reflexo do aumento do metabolismo da semente, tanto para resposta defensiva ao estresse hídrico quanto para o amadurecimento rápido da semente onde foram observados transcritos referentes a resposta oxidativa, controle hormonal, proteínas de reserva e produção de óleo.

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With the development and improvement of techniques for molecular studies and their subsequent application to the systematic, significant changes occurred in the classification of gasteroid fungi. The genus Morganella belongs to the family Lycoperdaceae, and is characterized mainly by lignicolous habit and presence of paracapilicium. Recent data demonstrate the discovery of new species for the group and the existence of a wide variety of species occurring in tropical ecosystems. However, the phylogenetic relationships of the genus, as well as the taxonomic classification, still require revisions to be better understood, the literature studies that address this issue are still very scarce. Thus, the objective of this study was to conduct studies of molecular phylogeny with species of the genus Morganella, to enhance understanding of the phylogeny of the group by including tropical species data. For this, the specimens used both for DNA extractions as for morphological review were obtained from Brazilian and foreign herbaria. For morphological analysis were observed characters relevant to the group's taxonomy. For phylogenetic analysis the Maximum Parsimony and Bayesian Analyzes were used, using the internal transcribed spacer (ITS) of nuclear ribosomal DNA. In phylogenetic analyzes, representatives from Morganella form a monophyletic clade with good support value and based on these results the genus should not be included as subgenus of Lycoperdon. The analysis indicated that M. pyriformis was not grouped with other representatives of Morganella, and therefore should not be included in the group as representative of Apioperdon subgenus because it is a Lycoperdon representative. Moreover, M. fuliginea, M. nuda, M. albostipitata, M. velutina, M. subincarnata are grouped with high support values within the genus Morganella. Morganella arenicola based on morphological and molecular studies does not aggregate in Morganella. Morganella nuda was grouped with M. fuliginea giving indications that can be treated as an intraspecific variation. The results of the analyzes favor to a better understanding of the species of Morganella. However, additional studies using a greater number of species, as well as other molecular markers are needed for a better understanding of the phylogenetic of Morganella.