Mestrado em Biotecnologia

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Alta pressão hidrostática como ferramenta de seleção direcionada em S. cerevisiae para produção de etanol 2g pela casca de coco verde
(Universidade Federal do Espírito Santo, 2019-03-11) Santos, Luiza Favarato; Fernandes, Patricia Machado Bueno; Santos, Alexandre Martins Costa; Torres, Fernando Araripe Gonçalves
The green coconut shell is a pollutant residue of the environment generated to tons in Brazil and that brings several environmental problems in its disposal. Therefore, using it for the production of renewable energy is an important sustainable solution for the mitigation of the environmental impact and for the generation of energy. Yeast Saccharomyces cerevisiae represents one of the microorganisms most used in the fermentation industries for the production of biofuels from lignocellulolytic residues. There are already on the market strains of this yeast capable of degrading lignocellulolytic residues, which have been developed to increase the yield of the process, making it feasible financially on an industrial scale. The high pressure hydrostatic (HHP) is a tool that simulates three types of different stresses in the yeast: the ethyl, the thermal and the oxidative, which are very common in fermentation dornas. In addition, studies have shown that fermentation occurs more rapidly when the yeast is under HHP. Therefore, HHP can be used as a targeted selection tool to obtain a strain with higher fermentation capacity and greater resistance to fermentation stresses, increasing the yield of the process. The aim of this work was to apply a selective pressure to the S. cerevisiae strain G2-104 using high hydrostatic pressure in order to obtain a strain with a higher fermentation capacity in green coconut shell culture medium, to increase the yield of the second generation ethanol production process.
Alterações na atividade fotossintética em folhas do mamoeiro ‘Golden’ infectadas com PMeV, detectadas por meio da fluorescência da clorofila a
(Universidade Federal do Espírito Santo, 2017-07-13) Hernández, Karla Verónica Alvarado; Silva, Diolina Moura; Moura, Andrea Bittencourt; Ventura, José Aires; Falqueto, Antelmo Ralph
Papaya (Carica papaya l.) is one of the most consumed fruits in the world due to its nutritional, pharmaceutical and use in the food and cosmetic industry. In Brazil, the second producer of papaya in the world, the production has been severely affected by meleira, disease which is caused by the complex of Papaya meleira virus (PMeV and PMeV2). In order to analyze the changes in photosynthetic activity in 'Golden' papaya leaves infected and not infected by PMeV, the experiment was carried out in which three groups of plants were evaluated: Control, without inoculation; Inoculated with sodium phosphate buffer (Treatment = ↓ [𝑃𝑀𝑒𝑉2]) and inoculated with sodium phosphate buffer and latex harvested from plants with meleira symptoms (Treatment = ↑[PMeV2]). The chlorophyll a fluorescence technique was used as a tool. There was a significant increase in quantum photochemical energy conversion in photosystem II (Y (II)) and an increase in the quantum yield of the regulated non-photochemical energy loss (Y (NPQ)) in the inoculated plants. At the same time, transient fluorescence curves showed greater efficiency in the reduction of the primary (Quinone A) and secondary (Quinone B) acceptors of electrons in photosystem II and greater efficiency of reduction of the plastoquinone pool and, consequently, greater efficiency in the reduction of acceptors of the Citb6f complex. The presence of two PMeV viruses and PMeV2 confirmed the presence of two PMeV viruses. A higher photochemical performance index (PItotal) was observed in the plants with the highest viral load from the 35th day after inoculation (DAI), and at 49 DAI, an increase in the reduction activity of photosystem I and PItotal was observed in plants with higher viral load and intermediate values in plants with lower viral load. These results suggest a defensive response of papaya cv. Golden to infection and confirm the presence of the two viruses, PMeV and PMeV2. Therefore, this technique proved to be a tool with great potential as a simple and fast method to study the interaction plant x pathogen and as the mechanisms of virus infection in plants and could also be used to detect the changes in photosynthetic activity in papaya infected with PMeV and PMeV2.
Alterações na parede celular e no metabolismo energético de Saccharomyces cerevisiae submetida à alta pressão hidrostática
(Universidade Federal do Espírito Santo, 2018-02-20) Carneiro, Tárcio; Fernandes, Patrícia Machado Bueno; Fernandes, Antonio Alberto Ribeiro; Santos, Alexandre Martins Costa; Zingali, Russolina Benedeta
Yeasts are one of the most important organisms in the processes of industrial biotechnology. Its great fermentative capacity and considered as a genetically safe organism, made this organism one of the first to be domesticated by the human being. Among its applications are not only its products generated from the fermentation, but also its cell itself, which moves a market ranging from organic yeast to yeast paste for human consumption. Understanding not only the fermentative process, but also the respiratory process are necessary. The production processes in which the yeasts are employed expose them to abiotic stresses of different types, such as temperature variation, pH, salinity and others. High hydrostatic pressure is now an important methodology in the study of its effects on the cells of Saccharomyces cerevisiae, acting as a model of stress. The understanding of this phenomenon on cells has helped not only to understand the processes of response to stress, but also its use as an important tool in industrial application. Atomic force microscopy, high performance liquid chromatography (HPLC) and microarray data were used to characterize the effects of high hydrostatic pressure on the cell wall of Saccharomyces cerevisiae and its energy metabolism. For the production of the experiments, BT0510 cells were submitted to high hydrostatic pressure (50 to 200 MPa) for 30 minutes and then analyzed by atomic force microscopy and in another experiment the cells were subjected to a pressure of 50MPa for 30 minutes and facing to agitation. The results showed the appearance of resistance bands in the cell wall at pressures of 100, 150 and 200 MPa, indicating possible fracture points, which leads to lesions that compromise its viability. At pressures of 50MPa the variation of the resistance of the cell wall resembles the control, which shows that this pressure is of sublethal character, not interfering so expressively in the cellular mortality. The response to 50 MPa treatment was then analyzed in the long term by HPLC, with collection points at 1, 2, 3, 4, 12, 36 and 60 hours after application of pressure. The results show high affinity with microarray generated data, where the cells increase the rate of glucose consumption in the fermentative phase (1 to 12 hours) as well as higher ethanol production. The cells also showed a greater efficiency in the ethanol metabolism which generated an increase in the cell mass in the cells treated with pressure. It was demonstrated that high hydrostatic 6 pressure has industrial application in cellular mass acquisition processes as in the production of biological yeast, thus showing the biotechnological capacity of the use of this stress mechanism and generating the deposit of the patent "Process for the increase of cellular mass in yeasts using high hydrostatic pressures ".
Análise da expressão dos genes relacionados à parede celular e a membrana plasmática de Saccharomyces cerevisiae em resposta a alta pressão hidrostática
(Universidade Federal do Espírito Santo, 2012-02-28) Souza, Diego Trindade de; Fernandes, Patrícia Machado Bueno; Masuda, Cláudio Akio; Guimarães, Marco César Cunegundes; Bravim, Fernanda
The yeast Saccharomyces cerevisiaehas received much attention for its applications in the biotechnology industry. However, during these processes, yeasts are exposed to many different types of stress. Knowing the mechanisms S. cerevisiaeuses to respond to these stresses is essential for obtaining higher yields in the industrial sector. High hydrostatic pressure (HHP) is a stress that exerts a broad effect on S. cerevisiae, interfering with cell membranes, cellular architecture, and various biochemical processes. In this study, we used the microarray technique to investigate the influence of HHP on genes related to the cell wall and plasma membrane. For this, cells from BT0605 strain were subjected to hydrostatic pressure of 50 MPa for 30 min, and then incubated at ambient pressure for 5, 10 and 15 min.The microarray results clearly demonstrated that of the genes involved in biogenesis of the cell wall, those which are related to protection were readily activated by high pressure, whilst the genes involved in cell wall structure required 5 minutes on the ambient pressure to be induced.
Análise da expressão temporal de genes relacionados ao metabolismo de Saccharomyces cerevisiae em resposta ao estresse por alta pressão hidrostática
(Universidade Federal do Espírito Santo, 2012-02-29) Silva, Lucas Ferreira da; Fernandes, Patrícia Machado Bueno; Masuda, Cláudio Akio; Louro, Iuri Drummond
Living organisms maintain a complex internal balance, which enables them to optimise their growth and metabolism. However, fluctuations in environmental conditions can affect this balance. The yeast Saccharomyces cerevisiae is a unicellular, eukaryote model organism. The biotechnological processes it is used for include the production of bread, cheese, alcohol, drugs and pharmaceuticals. From a physiological standpoint, all these processes affect the normal operation of the cell and it is extremely important to study the molecular mechanisms that provide protection against these stressful situations. One of the models used to study stress is high hydrostatic pressure (HHP), which is known to cause morphological and metabolic changes in yeast. Several studies have demonstrated that the expression pattern of genes from yeast submitted to high hydrostatic pressure shows a complex profile, including genes related to oxidative, osmotic, and heat protection.
Análise da fibrose do tecido adiposo de mulheres submetidas à abdominoplastia e cirurgia bariátrica
(Universidade Federal do Espírito Santo, 2014-10-28) Castro, Eduardo José Passamai de; Errera, Flávia Imbroisi Valle; Nogueira, Breno Valentim; Nahas, Fábio Xefran
Fibrosis is an exceeding accumulation of extracellular matrix, resulting from an unbalance between the synthesis and degradation of its components. It is associated with the metabolic alterations in the adipose tissue, however, its occurrence in the different deposits and clinical repercussions are not yet fully understood. The study aims at assessing fibrosis in the adipose tissue regarding the presence of obesity, location of the deposit [subcutaneous abdominal adipose tissue (SAAT) and the visceral one (VAT)] and its association with clinical variables. Samples of fat from SAAT and VAT were obtained from 21 women undergoing bariatric surgery (IMC>40kg/m²) and 25 VAT samples from the ones undergoing abdominoplasty (IMV<30kg/m²). The samples were processed for conventional histology. The red stain picrosirius was used to evaluate the total collagen fibers. The pictures obtained were analyzed in ADIPOSOFT®. The fibrosis percentage found in SAAT and VAT were analyzed through non-parametric static tests, using the value of p<0,05. SAAT amount present was bigger in women with obesity (p<0.0006). The fibroses between the deposits of SAAT and VAT was only observed among mulatto and black women with obesity (p<0,012). The amount of fibrosis in SAAT was not correlated to the variable clinics in women without obesity. However, with the ones undergoing bariatric surgery, it was observed correlations of fibrosis in SAAT with body mass index (BMI), glycated hemoglobin (A1c), LDL, triglycerides; and in VAT with the percentage of preoperative fat loss, percentage of total fat loss, percentage of pre lean mass, Basal Metabolic Rate (BMR) and Basal Energetic Consumption (BEC). The metabolic parameters and the anthropometric profile of the bariatric surgery were associated to the fibrosis in SAAT, while the post-surgery parameters were associated to the fibrosis in VAT.
Análise da influência de polimorfismos do gene CYP1B1 materno na idade gestacional de nascimento : uma correlação clínica e molecular
(Universidade Federal do Espírito Santo, 2017-04-20) Santos, Jéssica Aflávio dos; Louro, Iúri Drumond; Carvalho, Elizeu Fagundes de; Santos, Marcelo dos
Currently, both prematurity and births occurring in the late preterm and in the early term have been highlighted by their negative influence on infant mortality and morbidity. Several risk factors have been associated with decreased gestational age at birth. Environmental risk factors include cigarette smoking, excessive alcohol intake, ethnicity, weight and maternal age. Regarding genetic factors, genetic polymorphisms involved in oxidative stress and xenobiotics metabolism have been strongly associated with preterm birth. This may generate oxidative stress, considered a primary etiological factor for preterm birth because it can cause a compromised placenta. This is the case of the CYP1B1 gene, which also plays an important role in the estrogen synthesis, one of the most important hormones in pregnancy maintenance. The action of the enzyme CYP1B1 may result in the production of reactive and carcinogenic compounds due to the preferential hydroxylation of 17β-estradiol at the 4-hydroxy position. The polymorphisms rs10012, rs1056827, rs1056836 were selected for this work because they are associated with variations in this enzyme activity. Thus, the present study aims to investigate the possible influence of these polymorphisms, as well as clinical data and maternal life habits in gestational age at birth. For such, a prospective cohort study was carried out with pregnant women from the Santo Antônio de Jesus city, in State of Bahia, Brazil. Mothers were followed during gestation, delivery and puerperium between 2009 and 2016. Maternal characteristics and habits were obtained through standardized questionnaires. Genotyping was performed from blood samples from pregnant women with TaqMan® assays using the Real Time Polymerase Chain Reaction (PCR) technique. Pregnant women who delivered in the initial full-term period had higher mean maternal age and pre-gestational Body Mass Index (BMI) than the preterm and full-term periods. Pre-gestational BMI intervals, race, smoking, and alcoholic showed no significant association with gestational age at birth. A significant correlation was detected between the SNP rs1056836 and the maternal age and the pre-gestational BMI. None of the other analyzed variables showed a significant association with any of the other polymorphisms. Non direct association was detected between any of the analyzed polymorphisms and gestational age at birth. From the results obtained by this work, a possible relationship of the CYP1B1 gene with the gestational age at birth can not be ruled out. Subsequent studies should analyze the possible association of these polymorphisms with gestational age at birth in other ethnic groups, as well as carry out more extensive investigations including other genes and polymorphisms, as well as prematurity environmental risk factors.
Análise de ministr's em impressões digitais depositadas em suportes variados
(Universidade Federal do Espírito Santo, 2014-06-24) Vieira, Carolina Mayumi; Louro, Iúri Drumond; Paneto, Greiciane Gaburro; Rave, Cintia Fridman; Schuenck, Ricardo Pinto
The human fingerprint identification is a simple, fast and economical method. However, blurred prints deposited on rough surfaces constitute a challenge because of the loss of details. The development methods are insufficient to allow the analysis of the fingerprint, as observed points may be blurred or damaged. With the advances in DNA genotyping technology, small amounts of biological material such as a dozen cells can yield a complete DNA profile and be of great help in criminal investigations. In this context, fingerprints, previously only analyzed from the lophoscopic aspect, can now have a genetic approach. The use of MiniSTR 's combined with an increase in the number of PCR cycles can improve the detection of DNA in samples with degraded and/or limited amount of DNA. When investigating a crime scene, the forensic expert should keep in mind there is no hierarchy among evidence and every vestige should be analyzed considering its feasibility and effectiveness in generating proof.
Análise de mutações em formas recessivas de pacientes com osteogênese imperfeita do Espírito Santo : comparação de metodologias
(Universidade Federal do Espírito Santo, 2012-02-17) Quirino, Geise de Aguiar; Paula, Flavia de; Errera, Flavia Imbroisi Valle; Bortolini, Eliete Rabi
Osteogenesis Imperfecta (OI) is a genetic desease characterized by patient s bone fragility and deformity, in which severity ranges from a barely detectable connective tissue disorder to lethality in the perinatal period. The diversity of clinical variability in patients is caused by the different location or type of mutations in one of the ten genes related with the disease. This wide clinical variability difficults the perfect clinical diagnoses, due to that the use of molecular biology techniques becomes necessary to obtain a correct diagnoses and for genotype: phenotype correlation. One of the relevant genes associated with recessive forms of OI is the LEPRE-1 gene, responsible for encoding the prolyl 3 hidroxylase 1 protein. This protein and two others are components of the complex responsible for pro-collagen alfa 1 chains 3 prolyl hydroxylation. The target of this research was to analyze the LEPRE-1 gene in eight non consanguineous patients clinically diagnosed as severe Osteogenesis Imperfecta suggestive of autossomic recessive heritage by DNA sequencing of exons 1, 3, 5, 6 and 14 of the gene. In addition, the data obtained was used to analyze the efficiency of the SSCP technique by comparing the results between screening for mutations methodologies and gene sequencing methodologies. On exon 6, for instance, a mutation in one patient was found: a heterozygose base change (c.1087A>G / p.Lys363Glu), consequently, lysine was produced instead of glutamic acid. On the other exons, there was no mutation found on the patients chosen. All the results obtained in this research were compatible with datas generated by SSCP and suggest high efficient of SSCP technique for LEPRE-1 gene to recessive cases of Osteogenesis Imperfecta
Análise de um sistema de reabilitação para membros superiores utilizando ambiente de realidade virtual baseado em Kinect e sEMG
(Universidade Federal do Espírito Santo, 2016-02-29) Cardoso, Vivianne Flavia; Frizera Neto, Anselmo; Bastos Filho, Teodiano Freire; Nogueira, Breno Valentim; Ferreira, André; Andrade, Adriano de Oliveira
Impaired motor function appears as one of the most common symptoms stroke. When the upper limbs are affected, carrying out daily activities is compromised. The recent developments in the field of rehabilitation are exercises in virtual reality environment (RV). Another method used for rehabilitation of the upper limbs of patients after stroke is the EMG biofeedback. Through this biofeedback, the myoelectric signals from the muscle are converted into visual and audio information that allows the patient to control and regulate muscle activity. The objective of this work is to design and evaluate a new form of rehabilitation by biofeedback using RV and surface electromyography (sEMG) to complement conventional therapy upper limb post-stroke patients. The system was developed in accordance with the requirements in the design phase and design, considering the patient's functional limitations, residual skills, a motivating environment and ease of use. He is able to provide feedback of sEMG, the result (through the score in the game) and the angle described. The three games developed, aims to motivate the patient to perform elbow extension movements and meet the principles of motor learning. To develop and evaluate the system experiments were conducted with individuals without motor or neurological involvement. Analyzing the graph obtained by the myoelectric signal filtered and rectified is possible to observe a co-contraction of Bícpes Brachial (BB) and Trícpes Brachial (TB). When we analyze the results of feedback, the data show that and were not found significas differences (p = 0.9216) when playing with the dominant arm (BD) versus non-dominant (BND). However a significant difference is observed when comparing both arms versus BD (BD / BND) and p = 0.0003 vs. BND BD / BND, p = 0.0018. The results of the evaluations through the System Usability Scale (SUS), in the experiments of stage 2 (E2) was ± 81.4; SD ± 3.4 and stage 3 for participants (E3 / P) ± 82.5; SD ± 14.3 and rehabilitation professionals (E3 / PR) ± 81.1; SD ± 7.4). In Goal Attainment Scale (GAS), E2 was ± 73.3; SD ± 2.7; E3 / P ± 72.2; SD ± 6.1 and E3 / PR ± 73.5; SD ± 2.7. The results show good acceptance of the system by the users, and that the objectives expected during the use of the system have been achieved.
Análise do nível de estresse baseada em sinais de eletroencefalografia e de condutância da pele
(Universidade Federal do Espírito Santo, 2015-02-09) Pomer-Escher, Alexandre Geraldo; Bastos Filho, Teodiano Freire; Nogueira, Breno Valentim; Bravo, Eduardo Caicedo
The stress can affect everyone, independently of age, gender or ethnicity. The human body uses stress as an adaptive response in relations to various conditions, which require an adaptation of the body to face such a situation. Depending on the stressor stimulus, the stress is classified as physical, mental or emotional stress. However, the stress is not necessarily a bad thing or something pathological; in fact, it is a vital adaptation mechanism for the survival of the human species. The number of people who are affected negatively by stress has grown in last decades. Recent research highlights that in the United States about 60% to 90% of medical care were related in some way with stress. In Brazil, around 80% of the population have been affected by stress, and 30% of these are in the most critical phase. Nowadays, the main form of stress identification is still held by self-report questionnaire, thus, this research presents a methodology for stress analysis based on skin conductance and EEG signals. The parameters used for EEG are the asymmetry of alpha rhythm, and the ratio between the beta and alpha rhythms, both in the frontal and prefrontal cortex. For the EEG signal recording handheld device is used. It has electrodes that are specifically located on the positions according to the International 10/20 System (aF3, F3, F4, aF4). The participants are the Military Firefighters of the first Cia of Vitoria (Brazil), and three classes of emotional stimuli are used through the use of images belonging to the IAPS (International Affective Picture System) database, being called positive, calm and negatives classes. The accuracy of results through SVM (Support Vector Machine) classifier are 88.24% for positive stimuli class, 84.09% for calm class and 92.86% for negative class. Thus, this research presents a combination of parameters that can be measured with low-cost equipment, and provides conditions to differentiate stressful stimuli, and thereby providing indications that can be used to help the training of emergency care professionals.
Análise do padrão de ativação muscular de indivíduos hemiparéticos pós-AVC em marcha assistida por andador robótico
(Universidade Federal do Espírito Santo, 2015-02-20) Loterio, Flávia Aparecida; Frizera Neto, Anselmo; Bastos Filho, Teodiano Freire; Andrade, Adriano de Oliveira; Nogueira, Breno Valentim
Stroke is a leading cause of damages in the neuromuscular system. Robotic devices have been widely developed and studied in order to be used in gait assistance and gait training during the rehabilitation. The goal of this work is to evaluate the assisted gait by the AROW (Assistive Robotic Walker) in post stroke hemiparetic individuals through accelerometer signals and surface Electromyography (sEMG) analysis. The analyzed muscles are vastus medialis (VM), biceps femoris (BF), tibialis anterior (TA) and gastrocnemius medialis (GM). Furthermore, the evaluation methods GAS (Goal Attainment Scaling) and SUS (System Usability Scale) were used. Nine hemiparetic subjects participate of the experiments. The gait speed was decreased using the walker and, consequently, there were some changes in the duration of gait phases, for instance longer of support phase (p = 0.0174). The muscle activation pattern for the analyzed group did not show statistically significant difference (onset VM: p= 0.4999; offset VM: p= 0.5647; onset BF: p= 0.1186; offset BF: p= 0.7823; onset TA: p= 0.5833; offset TA: p= 0.8393; onset GM: p= 0.6077; offset GM: p= 0.1429).. However, assessing the muscle activation pattern individually, some benefic changes can be noted, for example, the reduced co-activation between tibialis anterior and gastrocnemius medial. The results of evaluations through GAS (54.8) and SUS (81.4) about the use of the AROW showed good acceptance among users, and the desired results during the use of the walker were achieved. The fast adaptation, easiness of use and feeling of safety when using the device are positive points that were obtained using the AROW.
Análise do perfil de expressão gênica diferencial de cepas de Saccharomyces cerevisiae laboratoriais e selvagens em diferentes estresses por ferramentas de bioinformática
(Universidade Federal do Espírito Santo, 2015-03-03) Xavier, Lorena Martins; Fernandes, Patrícia Machado Bueno; Fernandes, Antônio Alberto Ribeiro; Brandão, Rogélio Lopes; Von Zeidler, Sandra Lúcia Ventorin
Saccharomyces cerevisiae is an unicellular fungus used in many fermentation process since ancient times. This yeast is widely used in the ethanol production, thereby various strains are studied in order to develop a able to withstand the fermentation stress, which in turn will provide increased its production capacity. The high temperatures and increased ethanol concentration are common, but can be stress the yeast, thereby decreasing productivity. High hydrostatic pressure (HHP) appears as a good model for studying the fermentation stresses and can be used to increase the tolerance of the strains due to cross-protect mechanism, which is associated with an overall response to stress. The increase in reactive oxygen species (ROS) and low temperatures have pathways that fall into this general response too. The bioinformatics tools have proved of great assistance in the integration of regulatory pathways in order to improve understanding of responses in different experiments using cell metabolism studies. These can help the process of election able to make more durable and productive yeast. In this paper, we use data available in literature from microarray experiments of Saccharomyces cerevisiae strains under different stresses. We split the data into seven classes and gene expression patterns were analysed in Y440, BT0510 and BT0605 in different treatments of HHP stress, as well as, ethyl, thermal and oxidative stresses in DBY9434. We observed that the strains subjected to high hydrostatic pressure had a very similar gene expression profile, with the exception of the plasma membrane transport, signaling and cell polarization class. This category was studied in greater depth to understand the piezotolerance presented by the Y440 strain in HHP stress, thus we observed induction of glycerol-3-phosphate shutter, that generated oxidative stress in the cell. We also note the pentose phosphate pathway (PPP) induction, that which may have occurred as a response to such oxidative stress. Another feature observed was the sulfur amino acid synthesis induction too, a specific response of HHP stress. The responses to stresses to which the DBY9343 strain underwent were similar to each other, and we infer that the fact that the stresses selected for the analyzes have common stress response pathways between them. The Y440 strains, BT0510 and BT0605, subject to the same stress conditions, showed very similar responses also showing a certain standard of general common response to this stress. Bioinformatics tools were useful for the study of the modification of gene expression patterns in this stress group demonstrated that despite changes that occur in specific genes exists a cellular response model by modifying key routes for the maintenance of cell survival.
Análise do potencial da osteopontina e da Serpina1 como biomarcadores do carcinoma epidermóide de cabeça e pescoço
(Universidade Federal do Espírito Santo, 2017-02-22) Figuera Gómez, Celeny Milagros; Gouvêa, Sônia Alves; Paula, Flávia de; Figueiredo, Suely Gomes de; Balarini, Camille de Moura
Head and neck squamous cell carcinoma (HNSCC) is the most frequent histological type of head and neck cancers. Despite the numerous studies, a common set of biomarkers for HNSCC has not been established yet, evidencing the necessity to find molecules that facilitate the prognosis and early diagnosis of this disease. The objective of this research was to evaluate the Serpine1 and Osteopontin (OPN) as biomarkers in HNSCC because they are poorly evaluated molecules in this cancer. Biological samples and clinical-pathological data were collected from 40 patients with HNSCC and 11 healthy individuals. The detection of Serpina1 and OPN mRNA expression was performed by qPCR trials on tumor tissues and normal tissue samples. The evaluation of Serpina1 and OPN protein expression was performed with serum from both HNSCC patients and a control group by Western blotting. A significant increase in Serpina1 mRNA was observed in tumor tissue compared to normal tissue (p=0.0014) and in relation to different clinical-pathological characteristics evaluated (sex, smoking and smoking, staging and lymph node development). Serpina1 protein expression was also elevated in patients with HNSCC in relation to controls (p=0.0007) and in clinical-pathological characteristics evaluated. For OPN, no relevant significant differences were found for mRNA and protein except OPN protein expression, which was shown higher in the advanced stage (III/IV) (p=0.0181). Serpina1 has been shown as a potential biomarker of HNSCC for alterations found in tumor tissue and blood, which may facilitate its application in the development of the disease. The OPN should be better evaluated to establish the possible significant differences.
Análise dos parâmetros cinemáticos do plano sagital de joelho de pacientes com osteoartrose moderada durante a marcha livre e assistida por andador robótico
(Universidade Federal do Espírito Santo, 2013-05-03) Elias Neto, Arlindo; Frizera Neto, Anselmo; Bastos Filho, Teodiano Freire; Bó, Antônio Padilha Lanari; Fernandes, Antonio Alberto Ribeiro
abstract
Análise molecular do gene CRTAP através da técnica de PCR-SSCP-sequenciamento em pacientes com osteogênese imperfeita do Espírito Santo
(Universidade Federal do Espírito Santo, 2013-02-28) Almeida, Lorena Schneider; Paula, Flavia de; Errera, Flavia Imbroisi Valle; Maranduba, Carlos Magno da Costa
The Osteogenesis Imperfecta (OI) is a genetic disease characterized by structural defects of type I collagen protein or by reducing its biosynthesis causing decreased bone mass and predisposition to fractures and bone deformities. Approximately 90% of individuals with OI exhibit autosomal dominant inheritance caused by mutations in the genes COL1A1 and COL1A2. However, the number of genes linked to autosomal recessive forms of OI is increasing in the literature. The CRTAP gene was the second identified causing recessive inheritance OI. This gene has 6622 bp, seven exons and encodes a protein of 46.5 kDa. The CRTAP encoding the protein cartilage associated (CRTAP) which is part of the collagen 3-hydroxylation complex, responsible for post-translational modifications during the biosynthesis of collagen molecule. CRTAP mutations are related to severe and lethal form of the disease. The target of this research was evaluating the exons of CRTAP and its adjacent regions in OI patients from Espírito Santo thought the Single-Stranded Conformation Polymorphism (SSCP) screening of mutations and sequencing. We studied 24 patients with clinical diagnosis of OI from Hospital Infantil Nossa Senhora da Glória de Vitória, Brazil. The patients’ ages ranged from 2 to 16 years (median: 14.5). The sex proportion of the patients was 15 males and 9 females. Eleven patients have mild clinical symptoms of the disease, 5 show moderate symptoms and 9 were severe cases. The lethal OI cases were not obtained by methodological difficulties. We found the polymorphisms c.534C> T previously reported in exon 2 of the CRTAP gene in patients from sample. No pathogenic mutations were found in this study. The results of this study suggest that mutations in CRTAP are rare in ES population. These data may assist in developing more efficient methodological strategies for molecular diagnosis of OI.
Associação do carcinoma epidermóide oral com os polimorfismos MTHFR C677T, MTHFR A1298C e CBS 844ins68 na população do Espírito Santo
(Universidade Federal do Espírito Santo, 2013-09-18) Silva, Andressa Barboza da; Louro, Iúri Drumond; Von Zeidler, Sandra Lucia Ventorin; Pereira, Thiago de Melo Costa; Silva, Melissa de Freitas Cordeiro
Oral squamous cell carcinoma (OSCC) is a serious public health problem due to high mortality and rising incidence worldwide. Epidemiological studies suggest that OSCC susceptibility is mediated by interactions between genetic and environmental factors. Environmental factors include smoking, alcohol use and HPV infections. Among genetic factors, it has been observed that certain genetic variants may confer susceptibility to this disease. Studies suggest that genetic variants encoding enzymes involved in folate metabolism may modulate OSCC risk by altering DNA methylation patterns and genomic stability. Polymorphisms C677T and A1298C in the MTHFR gene that encodes the methylenetetrahydrofolate reductase enzyme and the CBS 844ins68 polymorphism in the CBS gene that encodes the Cystathionine beta-synthase enzyme may be associated with oral cancer susceptibility due to changes in catalytic activity.
Atividade antimicrobiana e antibiofilme da epigalocatequina galato em Staphylococcus aureus
(Universidade Federal do Espírito Santo, 2018-03-06) Knidel, Carina; Schuenck, Ricardo Pinto; Guimarães, Marco César Cunegundes; Silva, André Romero da
Bacterial infections are among the major public health problems and the emergence of antimicrobial resistant bacteria becomes increasingly common. Staphylococcus aureus is an opportunistic pathogen, can cause a variety of infections, both hospital and community. Epigallocatechin gallate (EGCG), a flavonoid present in the leaves of the plant of Camelia sinensis, has different biological activities, including antimicrobial potential. The objective of the present study was to evaluate the antimicrobial, in vitro and in vivo, and antibiofilm potentials of EGCG in clinical S. aureus isolates with different genetic backgrounds. Nine strains isolated from different infections and with different antimicrobial susceptibilities were used. The antimicrobial activity was performed by broth microdilution test to determine the minimum inhibitory concentration (MIC) and the time-kill curve test. The MIC results ranged from 7.81 to 62.5 μg/mL and bactericidal activity was observed with 4 times the MIC. The activity of antibiofilm was evaluated after incubation of the isolates in the presence and absence of EGCG. Sub-inhibitory concentrations were able to significantly inhibit S. aureus biofilm production. The largest reduction in biofilm production was 100% and the smallest reduction among 50-60%. Cytotoxicity assays showed that concentrations ≤ 62.5 μg/mL EGCG were non-cytotoxic to murine macrophages. Regarding the in vivo test with G. mellonella larvae, EGCG significantly reduced the mortality of larvae infected by this pathogen (P = 0.0005) in only one isolate. In general, EGCG showed efficacy in inhibiting the growth of different clinical isolates of S. aureus and exhibited a relevant property of antibiofilm. Thus, EGCG is a promising substance for the treatment of infections caused by S. aureus.
Autenticação de Ginseng brasileiro utilizando a técnica de High Resolution Melting (HRM)
(Universidade Federal do Espírito Santo, 2018-02-20) Almeida, Francine Alves Nogueira de; Paneto, Greiciane Gaburro; Ferreira, Marcia Flores da Silva; Moura Neto, Rodrigo
As the herbal industry grows, the authenticity of its products is a matter of consumer safety and cannot be neglected. Brazilian Ginseng, for example, refers to two distinct species, Pfaffia glomerata and Hebanthe eriantha. Despite the similarity between these two species, which hinders morphological identification, they have different chemical and pharmacological properties. In this project we proposed a High Resolution Melting technique (HRM) as a tool to discriminate and identify P. glomerata and H. eriantha species using a matK and rbcL gene region. For this purpose, six reference samples were obtained, three of each of the aforementioned species, and 60 (sixty) commercial samples, sold as Brazilian Ginseng, through physical or online purchase. The DNA is extracted by the CTAB method and kit. The primer designed were tested through amplification by conventional PCR and confirmed in polyacrylamide gel before standardization of the PCR amplificationHRM and finally the results were compared to sequencing. Three primers were developed for the matK gene, two for the HRM-matKD and HRM-matK, and one for the rbcL gene , HRM-rbcL . During the standardization, the influence of the annealing temperature and the concentration of the primer on the race result was noticed, however, the extraction method did not change the results. The analysis of HRM showed that the first HRM-matKD was the one that met the proposed objective presenting high sensitivity (92-93%) and specificity (100%) compared to the sequencing method. It was possible to validate the HRM technique using previously sequenced commercial samples, which allows us to state that the HRM technique can be used to discriminate H. eriantha and P. glomerata. HRM is a fast and low cost method, being a reliable tool for identification of Brazilian Ginseng species.
Avaliação da ação antimutagênica da ipriflavonacontra os danos induzidos por ciclofosfamida
(Universidade Federal do Espírito Santo, 2012-05-05) Delarmelina, Juliana Macedo; Batitucci, Maria do Carmo Pimentel; Paula, Flavia de; Marin-Morales, Maria Aparecida
Ipriflavone is a synthetic isoflavone derivative from daidzein and clinically prescribed for treating and preventing osteoporosis in postmenopausal women. We investigated the potential of this drug against the cytotoxic and mutagenic effects induced by cyclophosphamide (CPA) chemotherapy, using the micronucleus assay in bone marrow erythrocytes of Swiss albino mice (Mus musculus) in vivo. To evaluate their possible mechanisms of action, performed the evaluation of antioxidant activity by DPPH assay. For in vivo testing was carried out three protocols: pretreatment, simultaneous treatment and post treatment. The ipriflavone was evaluated in three different concentrations dissolved in DMSO (1,71; 8,57 e 42,85mg.kg-1 m.c) and administered by oral via. The bone marrow was collected for the evaluation of polycromatic erythrocytes (PCE) and the ratio PCE/(PCE+NCE) (polychromatic erythrocytes / polychromatic erythrocytes + normochromatic erythrocytes). For the DPPH test were assessed five concentrations of ipriflavone (500, 250, 150, 50 e 10μg.mLˉ¹) using DPPH solution (60μM). The results of in vivo tests show that the three concentrations of ipriflavone studied significantly reduced the frequency of MNPCEs induced by CPA, in the pre-treatment protocol and demonstrated the same effect at the concentrations of 1,71 e 42,85mg.kg-1 m.c in the post-treatment. However, simultaneous treatment did not reduce the frequency of MNPCE in any of the concentrations tested. In all protocols performed, the ratio PCE/(PCE+NCE) increased. There was variation between the genders in some of the experimental groups and the evaluation of antioxidant activity of ipriflavone showed no ability to donate hydrogens, suggesting that it acts through other mechanisms, such as inactivation of the enzyme activity of cytochrome P-450.

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