Doutorado em Biotecnologia RENORBIO

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Biomarcadores no câncer de mama e ovário : uma correlação entre alterações genéticas e aspectos histopatológicos
(Universidade Federal do Espírito Santo, 2013-02-04) Wolfgramm, Eldamária de Vargas; Louro, Iúri Drumond; Paula, Flávia de; Paneto, Greiciane Gaburro; Careta, Francisco de Paula; Silva, Melissa de Fretas Cordeiro
Breast and ovarian cancers are hormone-related diseases and polymorphisms in cancer genetic susceptibility genes involved in the production and metabolism of steroid hormones play an important role in the carcinogenesis of these tumors. Particularly, polymorphisms in short tandem repeat regions (STR) provide information about two events: microsatellite instability (MSI) and loss of heterozygosis (LOH). To check the presence of MSI and LOH in breast and ovarian cancers, 12 STR markers (CYP11, CYP19, UGT1A1, AR, ERα, ERβ, D5S345, D17S250, D10S197, D8S135, D3S1611 and D2S119) were analyzed in this study by Polymerase Chain Reaction (PCR) in 107 breast and 24 ovarian tumors. Single nucleotide polymorphisms (SNPs) in GSTP1 (A313G) and CYP17 (T27C) genes were also analyzed to estimate the frequency of susceptibility gene polymorphisms. In addition to the molecular analysis, an epidemiological study was conducted through the analysis of two oncology reference Hospital Pathology Service records in Espirito Santo, Brazil, during years 2001 to 2004 and 2009 to 2010. The epidemiological study detected 1,758 malignant breast and 119 ovarian tumors. Mean ages for malignant breast and ovarian tumors were 53.59 and 52.98 years, respectively. Among breast tumors, infiltrating ductal carcinoma was the most frequent malignant tumor and an increased tumor frequency in age group ≤ 35 years was observed for other malignant tumors of the breast in the time period 2009-2010, as compared to time period 2001-2004. The molecular study has shown that LOH is an event more frequently observed that MSI, in both breast and ovarian cancers. In breast carcinomas, the combination of STR markers showed that AR, CYP19 and ERβ, when analyzed together, were correlated with parameters of histological grade III, ER (estrogen receptor) negative tumors and PR (progesterone receptor) negative tumors. The combination of markers did not show significant results in ovarian tumors. Combination between STR markers and GSTP1 and CYP17 genotypes were performed, showing positive correlations among GSTP1 Ile/Ile genotype, AR+CYP19 (p=0.021) and AR+ERβ+CYP19 (p=0.036) alteration in PR negatives breast cancers, while CYP17 A1/A1 genotype was associated with AR+ERβ and AR+ERβ+CYP19 alterations in ER and PR negative breast tumors (p=0.039 to all combinations). We did not find associations between gene combinations and ovarian tumors. These data support the hypothesis that genes related to steroid metabolism are important in the characterization of breast cancer and that the analysis of a single polymorphism may not be enough to molecular characterize a tumor.
Aumento da atividade celulásica para hidrólise da casca de coco verde pela aplicação da alta pressão hidrostática
(Universidade Federal do Espírito Santo, 2013-04-01) Albuquerque, Érica Dutra; Fernandes, Antonio Alberto Ribeiro; Fernandes, Patricia Machado Bueno; Marco, Janice Lisboa De; Torres, Fernando Araripe Gonçalves; Castro, Eustáqio Vinícius; Gomes, Daniel Cláudio de Oliveira
The use of cellulosic biomass for cellulosic ethanol production has been a serious option for the production of renewable fuels. The main focus in the cellulosic ethanol production is to increase the hydrolysis of these cellulosic materials in order to produce products with a low-molecular weight, such as hexoses.The high costs involved with this process due to the use of cellulases and the biomass pretreatment, causes the main problem in an economically viable large-scale production of bioethanol. Even though enzymatic disruption of cellulose has been considered the best environmental practice, cellulase is the most expensive step during cellulosic ethanol production. Another problem is the inhibition of the cellulase complex by reducing-sugars such as cellobiose and glucose. Significant cost-reduction becomes needed in order to enhance viable commercial use of cellulase hydrolysis technology. Using coconut husk as a biomass substrate, we applied two approaches: firstly, we isolated fungi from coconut husks. We found that their cellulases outperforme the cellulases of the industry standards on the hydrolysis of coconut husk. Secondly, we employed High Hydrostatic Pressure (HHP) to the cellulases in order to improve the enzymatic performance. After the HHP treatment, we observed an increase in coconut husk hydrolysis by a factor of 2 as a result of an increase of the βglucosidase, endoglucanase and total cellulase activities. The effect of HHP is able to break the hydrogen bonds working down to the scales of the coconut fibers. Further, the inhibition of β-glucosidases caused by glucose was removed under HHP, the βglucosidases achivied higher activities in the presence of 55 mM of glucose as a result of changes in the conformation of this cellulase on HHP. Using bioinformatics tools to analyse structures of β-glucosidase and endoglucanase, we found hydrophobic aminoacids and glycosylation regions in the cavities; in the active sites; and on β-sheets structures. While HHP affects the cavities of the proteins, the carbohydrates can bind both to their side chains of hydrophobic amino acids as to glycosylation regions, leading to an increase of the activities and a stabilization of the protein. HHP to value of 300 MPa exposes the internal apolar parts of the coconut fiber (CEF) and favors the binding of hydrophobic aminoacids of the cellulase to these parts of the fibers, as well as to glucose. The increase of the cellulase hydrolysis under HHP can be caused by a factor that works like a CBM (Carbohydrate Binding Module) that approaches the substrate at the active site. Thus, employing both the 21 technologies HHP as well as substrate specific fungi can potentially make bioethanol production an economic viable and energy-efficient process, especially regarding the improvement of β-glucosidase activities.
Análise da expressão de genes relacionados à adipogênese e à inflamação em tecido adiposo de mulheres com obesidade grau III
(Universidade Federal do Espírito Santo, 2013-04-29) Dasilio, Karine Lourenzone de Araujo; Errera, Flavia Imbroisi Valle; Paula, Flávia de; Louro, Iuri Drumond; Nogueira, Breno Valetim; Guiomarães, Marco Cesar Cunegundes; Costalonga, Everlayny Fiorot; Pereira, Thiago de Melo Costa
Obesity is a chronic condition defined as abnormal or excessive fat accumulation that impairs health. Overweight is an important risk factor for mortality and is a major risk factor for noncommunicable diseases, such as cardiovascular disease, impaired glucose tolerance (IGT) and type 2 diabetes mellitus (T2DM). Deposits of white adipose tissue (WAT), such as subcutaneous (SAT) and visceral (VAT), are biologically distinct, consequently, exhibited different biological effects on the health. WAT produces and secretes a variety of adipokines, such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), adiponectin (ADIPOQ), secreted frizzled-related protein 1 (SFRP1) and vascular endothelial growth factor (VEGFA), that are important in several cell functions, including angiogenesis, inflammation and adipogenesis. Alterations in adipokines expression might contribute to the onset of co-morbidities of obesity. The activation of wingless-type (WNT)/β-catenin pathway inhibits of adipogenesis via the blockade of CCAAT/enhancer binding protein alpha (CEBPA) and the peroxisome proliferator-activated receptor gamma (PPARG), which are essential transcriptional regulators of adipocyte differentiation. Evidence showed that non-fibrillar collagen XVIII (COL18A1), a protein of the extracellular matrix, has been shown to be active in adipogenesis. The aim of this study was, through quantitative Polymerase Chain Reaction, evaluate the expression of genes involved in adipogenesis and inflammation in SAT and VAT of women with severe obesity. The main results showed that i) the expression of the gene CEBPA in SAT is higher than in VAT of women with obesity ii) SAT CEBPA gene expression is higher in obese women with T2DM/IGT than in obese women with normal tolerance glucose tolerance (NGT), iii) the expression of SFRP1 gene in VAT was lower in women with T2D/IGT than those with NGT, iv) VAT COL18A1 gene expression was associated with better HDL levels and tolerance glucose. These results showed that the expression of genes involved in adipogenesis and inflammation differs in distinct WAT deposits and the expression of these genes were correlated with the metabolic parameters in obese individuals. This work contributes to a better understanding of the expression of genes related to these biological processes in SAT and VAT of patients with severe obesity.
Avaliação e caracterização de insumos bioativos da aroeira (Schinus terebinthifolius Raddi) com potencial econômico para o desenvolvimento tecnológico de bioprodutos
(Universidade Federal do Espírito Santo, 2013-06-20) Sales, Maria Diana Cerqueira; Fernandes, Patricia Machado Bueno; Ventura, José Aires; Costa, Hélcio; Kuster, Ricardo Machado; Meira, Débora Dummer; França, Hildegardo Seibert
Aroeira (Schinus terebinthifolius Raddi) is an important plant mainly due its industrial application in the food industry, cosmetics and pharmaceutics. Since society has been exigent to choice qualified products, in this work we aimed identification and evaluation of bioactive products, such as mother tinctures (MT), extracts (Eb) and essential oils (EO) from leaves and fruits of aroeira genotypes. The TM were obtained from the leaves of plants hermaphrodite (TMArFH), female (TMArFF), and male (TMArFM) and fruits from hermaphrodite plants (TMArFrH) and two samples of female plants (and TMArFrF1 TMArFrF2), were prepared from the plant extract (with natural adjuvant) by maceration. OE from leaves (F01-F04) and fruits (the FR01- FR07), were obtained from the hydrodistillation. Analytical methods were used to perform physic-chemical analyses of MT and chemistry compounds identification were carried out by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography-Mass spectrometry (GC/MS). The antioxidant activity of the different MT was evaluated using the stable free radical DPPH (1, 1- diphenyl-2- picryl-hydrazyl) method and, evaluation of antifungal efficiency of TM was performed by the agar diffusion method, for the fungi Fusarium guttiforme and Chalara paradoxa, pineapple pathogens. The physico-chemical analysis showed average values of dry residue (1.41 ± 0.13%), alcohol content (67% ± 0.78), pH (5.6 ± 0.78) and density (0.898 g. mL-1 ± 0.98), within the technical specifications, indicating quality in the process and specifications to obtaining the TM. The chromatograms showed the presence of phenolic groups, especially, isolation of two important biflavonoids, agathisflavona and tetrahidroamentoflavona. Monoterpenes predominated, and the sample FR01, had the highest percentage of limonene (36.85%), which represented the highest relative content (70.85%). Regarding the antioxidant activity (AA), the TMArFH (250 µg/mL), showed better availability for uptake of DPPH (AA= 59.09%). In the antifungal test both TMArFH as tebuconazole fungicide at 0.1%, showed strong inhibition of the mycelial growth for F. guttiforme (PI= 82%) and a significant efficiency for C. paradoxa (PI= 42. 86%). This is the first report with the use of MT from leaves and fruits of S. terebinthifolius with potential feedstock for the development of bioproducts with fungicidal activity for control plant pathogens.
Análise da expressão de genes relacionados à baixa densidade mineral óssea : avaliação prognóstica e de conduta terapêutica para osteoporose
(Universidade Federal do Espírito Santo, 2013-11-22) Souza, Leticia Soncini de; Bastos Filho, Teodiano Freire; Silva, Ian Victor; Nogueira, Breno Valentim; Guimarães, Marco César Cunegundes; Houzel, Jean Christophe; Pires, Rita Gomes Wanderley
Since that assuming the knowledge of the regulatory mechanisms of bone formation and resorption is crucial in the search for alternative therapies in diseases such as osteoporosis, molecular biology emerges as interesting tool and essential to achieving this goal. Among these genetic markers, gene polymorphisms associated with estrogen receptor alpha (ERα) and the gene Apolipoprotein " E" (ApoE) have received increased attention in recent years. The aim of this study was to analyze the expression of polymorphisms PvuII (CT nt -397) and XbaI (GA nt -351) gene present in REα and polymorphisms in the gene ApoE (HhaI - ε2 , ε3 and ε4) in populations of postmenopausal women, associating these gene alterations, and their clinical and biochemical profiles with osteoporosis. After analyzing the results obtained, the PvuII SNP in the gene of ERα is related to low BMD, and this effect is most noticeable in women with advanced age. The P allele, however, correlates strongly with high BMD (p < 0.05) in the whole population studied and reproduced when analyzing the population aged over 65 years, suggesting a protective role in the loss of bone mineral. In XbaII SNP, of same gene, a significant association of allele x at concentrations of triglycerides and total lipids and dependence on patient age and Body Mass Index (BMI) was observed. On the other hand, for SNP HhaI, ApoE gene, the E2 allele may be associated as a risk factor for low BMD, and E3 allele may be associated as a protective factor in relation to BMD. These results contribute to a better understanding of the expression of genes related to osteoporosis and can provide information for better prognostic determination of the disease as well as rationalization of therapeutic conduct to be chosen, providing a better quality of life for postmenopausal patients.
Efeitos dos hormônios sexuais femininos e da apolipoproteína E na expressão de biomarcadores característicos da Doença de Alzheimer em animais
(Universidade Federal do Espírito Santo, 2013-11-22) Pedrosa, Diego França; Bastos Filho, Teodiano Freire; Silva, Ian Victor; Nogueira, Breno Valentim; Guimarães, Marco Cesar Cunegundes; Silva, Cristina Martins e; Houzel, Jean Christophe
Alzheimer's disease (AD) is a progressive neurodegenerative disease that compromises elements of the central nervous system, commonly associated to memory loss and dementia. There has been observed that AD’s patients brains exhibit extracellula plaques that consist in deposits of beta-amyloid (Aβ) peptide. Also, intracellular neurofibrillary tangles, which can be associated to intermediate filaments, appear mainly in the hippocampus and cerebral cortex of these patients. Anamnesis and molecular studies have suggested the presence of genetic elements involved in this pathophysiology, such as APOE gene, beta-amyloid precursor protein (APP) and TAU protein. However, some other comorbidity (high glycemic and lipid levels) may also contribute for the disease onset. Moreover, it is known that the disease incidence increases rapidly with advancing age and AD affects proportionately more women than men. It is believed that the decline in gonadal hormone levels in postmenopausal women may contribute to the observed difference between the sexes as long as it is also associated to many decline of body functions described above.
Protocolos e técnicas de análise de sinais sEMG aplicados à avaliação motora e robótica
(Universidade Federal do Espírito Santo, 2013-12-16) Vela, Jhon Freddy Sarmiento; Silva, Ian Victor; Bastos Filho, Teodiano Freire; Fernandes, Antonio Alberto Ribeiro; Nogueira, Breno Valentim; Guimarães, Marco César Cunegundes; Andrade, Adriano
Technological advances in the last decade opened up the field for the development of information processing systems with high capacity of data storage. These advances in health have evolved in the development of devices for applications in Bioengineering and Biomedical Engineering, supporting the understanding of the physiological behavior, diagnosis, monitoring, treatment and control of various biological changes. Along with technological advances, the amount and complexity of information is increasing, compared to its usefulness and understanding, representing, for different areas of knowledge, a challenge to find viable alternatives for using the attributes of biological systems in the development of new technologies directed to improve the quality of life of human beings. Currently, the development of noninvasive protocols for capturing bioelectric signals are becoming a viable option for the diagnosis of myopathies, motor rehabilitation, biomechanical analysis, development of Human-Machine Interface, and autonomous control of robotic devices for people with severe motor disabilities among other applications. In all cases, the support of computational techniques, such as digital signal processing (DSP), and new algorithms based on artificial intelligence, has opened the opportunity to develop classification techniques for recognizing patterns which can be applied in biotechnology for health. This doctoral thesis develops protocols and techniques for analysis of sEMG signals, consisting of "instructed delay tasks", applied to the motor assessment and rehabilitation estrategies, involving analysis of inclusion-exclusion criteria for clinical history, control variables in experimental environment, capture, acquisition and processing of sEMG signal, digital group, filtering, segmentation, feature selection, classification and pattern recognition. Biotechnological applications with sEMG signals present a quantitative experimental approach in the form of case studies. The first case study is centered on three acquisition protocols for evaluation of proprioceptive knee, control of a robotic wheelchair for people with severe motor disabilities, and manipulation of a mobile robot for children with cognitive and motor disability, using a hybrid sensor (inclination + sEMG), which is a patent derivate of this thesis. The second case study, develops a protocol for acquisition of sEMG signals in order, to support the diagnosis of fibromyalgia using algorithms for evaluation of muscle fatigue in time domain (ARV, RMS) and frequency domain (MNF, MDF, AIF), with 30%, 60% and 80% of MVC. The third case study, develops a protocol for the acquisition of sEMG signals with low density and low level of muscle contraction, with control of the rest, for the recognition of different hand gestures in healthy and amputees, evaluating 14 characteristics , 8 in time domain, and 5 in frequency domain and Fractal Dimension (FD), with several of their combinations, which were classified with computational techniques of artificial intelligence, such as fuzzy logic (FL) and artificial neural networks of MLP type. The results for the first case study, has demonstrated the usefulness of threshold predetermination as RMS and slope, acquired with the hybrid sensor (inclination + sEMG), improving the accuracity sense of positioning in proprioceptive analysis of the knee compared to a commercial electrogoniometer in combination with sEMG signal. The hybrid sensor also was applied to the control of a robotic wheelchair, using head movements for self-displacement of persons with tetraplegia, as well as autonomous manipulation of a mobile robot by people with cognitive and motor disabilities, which was obtained with training, whose performance in interacting with the robot was evaluated by GAS index. In the second case study, the results obtained for assessment of fatigue in people with fibromyalgia (FM)have indicated a relationship between increasing load and muscle pain, especially with 80% of MVC. The linear regression of algorithms RMS, ARV and MNF havshown in both the inclination (α ) and intercept (β) an expected trend in the control group, with positive linear relationship to characteristics in the time domain and negative characteristics to the frequency domain, with 60% MVC, and 60% of isometric segment of sEMG signal, which were obtained with 20 isotonic contractions during flexion-extension of biceps braquii (RMS α = 1.1319, β = 275 706; MNF α = -0470, β = 91 482). In the case of volunteers with FM, the N3 voluntary presented a behavior with the highest expected trend of muscular fatigue at 80% MVC and 60% of isometric segment, obtained during isotonic movement of biceps braquii (RMS α = 5.92 β = 113.33; MNF α = β = -1.21 96.96). Finally, the third case study, identified, with the MLP classifier, a success rate of 94.9% for six movements of individual fingers, including rest (category A), and 97.5% of success rate for seven movements, including: fingers, wrist and grip (category B), both cases, with a combination of features RMS, WL, MAV and ZC. On the other hand, the results obtained by amputee volunteers showed better results with features in time domain, compared to fractal dimension (DF), with success rates of 93.9% using combination RMS, WL and MAV characteristics for category A, and 95.4% of success rate with combination of RMS, WL, MAV and ZC in category B.
Estudo genético e de instabilidade genômica da doença Alzheimer em pacientes de Vitória-ES
(Universidade Federal do Espírito Santo, 2014-02-20) Belcavello, Luciano; Batitucci, Maria do Carmo Pimentel; Paula, Flavia de; Oliveira, João Ricardo Mendes de; Nogueira, Breno Valentim; Morelato, Renato Lirio; Santos, Eldamária de Vargas Wolfgramm dos
abstract
Desenvolvimento de processo para a purificação de bromelina a partir de resíduos de abacaxizeiro (Ananas comosus var. comosus) cv. Vitória
(Universidade Federal do Espírito Santo, 2014-02-20) Costa, Helber Barcellos da; Ventura, José Aires; Kuster, Ricardo Machado; França, Hildegardo Seibert; Costa, Hélcio; Romão, Wanderson
abstract
Avaliação de marcadores moleculares relacionados ao prognóstico de pacientes com carcinoma epidermóide de cavidade oral e orofaringe
(Universidade Federal do Espírito Santo, 2014-08-29) Santos, Marcelo dos; Conforti, Adriana Madeira Álvares da Silva; Louro, Iuri Drumond; Paula, Flávia de; Nogueira, Breno Valentim; Guimarães, Marco Cesar Cunegundes; Tajara, Eloiza Helena
Head and neck cancer is the fourth in incidence and the fifth in mortality among the most frequent malignancies worldwide. For 2014, there has been estimated over 15 thousand new oral and oropharynx cancers in Brasil. Similar to other tumors, oral and oropharynx cancer is a multifactorial disease, caused by multiple environmental and genetic factors, involving alterations in molecular pathways and cellular homeostasis. The investigation of molecular markers involved in this process has been the object of my studies, especially because in spite of great advances in the molecular aspects of cancer, little progress has been made in the clinical outcome during the last decades. It is known that tumor progression depends on cellular aquisition of competences, such as apoptosis evasion, cell proliferation disregulation, angiogenic activation, cellular adaptation to hipoxia, cell survival mechanism activation and hypoxia dependent epigenetic changes. Therefore, the present work had the purpose to evaluate the potential of proteins FAS, FASL, FGFR4, LEPR, HIF1-a, NDRG1 and JMJD1a, as well as polymorphisms FGFR4 Gly388Arg and LEPR Gln223Arg, as putative molecular markers for clinicopathological tumor features or oral and oropharynx squamous cell carcinoma. Our results show that HIF1-1 expression was associated with local disease relapse and local disease-free survival in patients who undertook pos-operative radiotherapy and were also related to tumor vascular microdensity. In addition, NDRG1 expression was different in tumor tissue and non tumoral margins, but also showing an association with disease survival. FGFR4 polymorphism and expression showed a relation with death and disease survival. However, FGFR4 expression alone showed an association with lymph node metastasis and relapse. FGFR4 profile showed a relation with disease survival. FAS/FASL expression showed a correlation with death and its proposed profise was related with disease survival. LEPR Gln223Arg polymorphism was realted with disease free survival and disease specific survival, whereas LEPR expression was realted with lymph node metastasis. JMJD1A nuclear and cytoplasmic expression were related with lymph node metastasis. However, only nuclear expression was related with relapse and disease survival. In conclusion, our results suggest that proteins ans polymorphisms can be used as molecular markers to help predict prognosis in oral and oropharynx squamous cell carcinoma.
Osteoporose : uma análise ultraestrutural do tecido ósseo em camundongos C57 e apoeko ovariotectomizados
(Universidade Federal do Espírito Santo, 2014-09-26) Siqueira, Daniel de; Rangel, Letícia Batista Azevedo; Silva, Ian Victor; Nogueira, Breno Valentim; Pacheco, Marcos da Silva
It is currently observed an increase in the incidence of osteometabolic diseases, particularly osteoporosis. This disease is caused by an imbalance between bone formation and bone resorption, closely linked to aging. In osteoporosis, it is observed that the predominance of bone resorption leads to a lower bone mineral density (BMD). It is believed that decrease in BMD is the main risk factor for bone breaking observed in these patients. Although low BMD represents the main risk factor for osteoporosis, this disease is multifactorial. Another remarkable feature in osteoporosis is its prevalence in postmenopausal women. It is now known that estrogen is one of the major factors responsible for the formation of bone tissue and its mineralization. Therefore, its deficiency at menopause results in a higher incidence of the disease in this population. Nevertheless studies in humans have contributed immensely to the understanding of the disease, the limitations imposed by the techniques to be used prevents the investigation of some mechanisms associated with osteoporosis. To determine some intrinsic mechanisms of the disease, therefore, animal models that mimic the postmenopausal osteoporosis are commonly used. In this study, we used ovariectomized female mice (OVX) as a model for postmenopausal osteoporosis, aiming to study phenomena associated with the bone tissue ultrastructure under conditions that can be of great importance for the treatment of the disease. For this purpose, were used a combination of histological and biochemical analyses as well as electronic microscopy, besides the gold standard in the diagnosis of osteoporosis: Dual X-ray Absorptiometry (DXA). The drop in circulating estrogen led to profound changes in different markers of bone formation and bone resorption, decreased BMD, and the presence of microfractures in bone tissue. From this characterization, were conducted two parallel studies, despite complementary, on the role of vitamin K - molecule believed to be essential for bone mineralization - and apolipoprotein E, a molecule that is also associated to increased risk of osteoporosis. Results obtained throughout this study demonstrated that supplementation with vitamin K in OVX animals led to profund changes in the ultrastructure of bone tissue (determined by analysis of scanning electronic microscopy). However, supplementation with vitamin K did not lead to such deep changes in biomarkers of the disease. In contrast, the loss of ApoE in genetically modified animals (APOEKO) significantly changed several serum biomarkers for the diagnosis of osteoporosis and the ultrastructure of tissue in OVX mice. Taken together, our results indicate - through a combination of ultrastructural analyses on bone tissue and measurement of biomarkers - that vitamin K affects mainly microfractures in OVX mice while the absence of the ApoE gene led to a decrease in BMD. Therefore, even if the axis estrogen-ApoE-vitamin K is well defined in the context of the pathogenesis of osteoporosis, our results pointed to different effects of these molecules on bone metabolism.
Influência de polimorfismos nos genes FcγRIIa, CD209, VDR, TNF-α, IL-4, IL-6 e INF-γ na persistência de sintomas clínicos da dengue na fase de covalescença
(Universidade Federal do Espírito Santo, 2015-02-13) Dettogni, Raquel Spinassé; Louro, Iuri Drumond; Paula, Flávia de; Silva, Melissa de Freitas Cordeiro; Conforti, Adriana Madeira Álvares da Silva; Carvalho, Elizeu Fagundes de
Differences in host susceptibility to infection, in the severity and permanence of the clinical picture of disease can be attributed, in part, to variations in the immune response. These variations are associated with single nucleotide polymorphisms (SNPs). As a previous study, the characterization of the general population of the Espírito Santo (ES)-Brazil and of a subpopulation of the state, of pomeranian origin, was performed as the SNPs -131 H/R, -336 A/G, TaqI, -308 A/G, -590 T/C, -174 G/C and +874 A/T in FcγRIIa, CD209, VDR, TNF-α, IL-4, IL-6 and IFN-γ genes, respectively. One hundred individuals of the Grande Vitória represented the general population of ES and 59 individuals of Santa Maria de Jetibá represented the population of Pomeranian origin. As the acute phase of dengue is well characterized, this study aimed to expand the knowledge of the phase of convalescence. Ninety-six individuals diagnosed with symptomatic dengue in late 2012 and early 2013, in ES, were followed for 60 days from the onset of symptoms by completing a clinical and epidemiological questionnaire in four interviews. The persistence of 37 clinical symptoms of dengue was assessed. To analyze the influence of the genetics of the host immune system in the persistence of clinical symptoms of dengue in the convalescent phase, the association between seven SNPs, for which the population of ES was characterized, and the persistence of symptoms was determined. Genomic DNA of study participants was extracted from peripheral blood and genotyping of SNPs was performed by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP). The genotype frequencies of all SNPs were found in Hardy-Weinberg equilibrium (HWE), except for the SNP in the IL-6 gene. There was no statistically significant difference in genotype frequencies of SNPs in FcγRIIa, CD209, VDR, TNF-α and IL-4 genes between the two populations. A statistically significant difference was found between the two populations in the genotypic distributions of SNPs in IL-6 (p = 0.03) and INF-γ (p = 0.007) genes. Thirty and sixty days after the onset of symptoms, 38.5% and 11.5% of symptomatic patients with dengue reported having at least one clinical symptom of dengue, respectively. Symptoms among, the most persistent were related to fatigue syndrome as myalgia, arthralgia, asthenia and malaise, myalgia being the most frequent. The persistence of symptoms at 30 days was associated with female gender (p = 0.044) and persistent constitutional symptoms was associated with secondary dengue (p = 0.041). FcγRIIa gene SNP, was associated with persistent symptoms at 30 days in the subgroup of patients with secondary dengue (p = 0.046), and presence of the H allele was associated with non-persistence of symptoms (p = 0.014). The presence of the A allele in the TNF-α gene SNP was associated with non-persistence of symptoms in the subgroup of patients with secondary dengue (p = 0.025), with the GG genotype associated with persistent neurological, psychological and behavioral symptoms in 30 days (p = 0.038). The presence of the C allele in the IL-6 gene SNP was associated with persistent dermatological symptoms at 30 days (p = 0.005). Genetic profiling of these SNPs may favor the establishment of immunogenetic markers associated with the convalescent infection phase by the dengue virus (DENV).
Investigação de mutações nos genes LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 e SERPINF1 causadoras da osteogênese imperfeita recessiva
(Universidade Federal do Espírito Santo, 2015-02-13) Furtado, Clara Fernanda Barbirato; Paula, Flávia de; Cordeiro-Silva, Melissa de Freitas; Conforti, Adriana Madeira Álvares da Silva; Carvalho, Elizeu Fagundes de; Batitucci, Maria do Carmo Pimentel
Osteogenesis Imperfecta (OI) is a clinically and genetically heterogeneous disease predominantly characterized by bone fragility and deformity and recurrent fractures. Most cases of OI result of autosomal dominant mutations in COL1A1 and COL1A2 genes that encode the chains forming type I collagen, the main protein in bones. In the past few years, an increasing number of cases due to recessive mutations has been reported in genes associated with the biosynthesis of type I collagen or to the formation and bone mineralization, such as LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1. Mutations in these genes, in general, lead to the development of severe and lethal OI phenotypes. In this work, LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1 of 25 OI patients were analyzed using SSCP and automated sequencing. Altogether, 29 genetic variations were detected, mutations and polymorphisms. Among the eleven variants found in LEPRE1 gene, there are the already well described c.1080 + 1G> T and the potentially deleterious mutations c.2024G> A / p.Lys363Glu and c.1501C> T / p.Arg501Trp . In FKBP10 gene, the previously described duplication c.831dupC, and c.1546G>A / p.Leu516Phe, predicted to be disease causing, were detected. It was observed that FKBP10 and LEPRE1 contain the most important mutations found in the patients studied in this work and it is suggested that LEPRE1 and FKBP10 should be preferably analyzed in studies of screening and identification of mutations in patients with OI. To date, there are no reports of mutations in LEPRE1, CRTAP, PPIB, FKBP10, SERPINH1 and SERPINF1 genes in Brazilian patients and this study provides new information on the genetic aspects of recessive OI.
Envolvimento de microRNAs na interação Carica papaya L. e Papaya meleira vírus com potencial biotecnológico
(Universidade Federal do Espírito Santo, 2015-02-26) Abreu, Paolla Mendes do Vale de; Fernandes, Patrícia Machado Bueno; Zanettini, Maria Helena Bodanese; Silva, Maite Vaslin de Freitas; Rodrigues, Silas Pessini; Fernandes, Antônio Alberto Ribeiro
Carica papaya L. is one of the most cultivated and consumed fruits in tropical and subtropical regions worldwide. Brazil is among the largest producers and exporters of papaya fruit. The pre-harvest diseases of papaya plants are the main limitation for fruit production. Papaya sticky disease is caused by the Papaya meleira virus (PMeV). It is a commercially important pathology in papaya culture potentially causing the complete loss of fruit production. Despite of this, little is known about the papaya interaction and response mechanisms against PMeV and there is not a papaya variety resistant to the virus. It is known that papaya 20S proteasome subunit levels of increase during PMeV infection, suggesting that proteolysis is an important feature of the plant defense response mechanisms. To date, 10,598 plant microRNAs have been identified in the Plant miRNAs Database (name of the DB), but only two microRNAs, miR162 and miR403, are from papaya. In this study, plant microRNA sequences were used to search for putative microRNAs in the papaya genome. A total of 462 microRNAs, representing 72 microRNA families, were predicted to occur in papaya. Out of these, the expression of 11 microRNAs, whose targets are known to be involved in 20S and 26S proteasomal degradation and in other stress response pathways, was estimated using real-time PCR, comparing healthy and infected papaya leaf tissues. The expression of miRNAs involved in proteasomal degradation increased in response to very low levels of PMeV titre and decreased as the viral titre increased. In contrast, biotic stress-related miRNAs levels decreased in papaya tissues infected with low virus titre and increased at high PMeV levels. Corroborating this results, analysed target genes for this miRNAs had their expression modulated in a dependent manner. With the results, some miRNAs were identified as relevant to the biotechnological application. This study represents a comprehensive prediction of miRNAs in papaya. The data presented here might help to complement the available molecular and genomic tools for the study of papaya. The differential expression of specific miRNAs and the modulation of their target genes will be helpful for understanding the particular interaction of PMeV and papaya responsible of disease development.
Mecanismos envolvidos na progressão do câncer de ovário : 1) papel de NAC1 e BCL6 na regulação da expressão gênica; 2) efeito da cisplatina no fenótipo de células-tronco tumorais, migração e quimiorresistência
(Universidade Federal do Espírito Santo, 2015-04-27) Herlinger, Alice Laschuk; Rangel, Letícia Batista Azevedo; Pacheco, Marcos da Silva; Pires, Rita Gomes Wanderley; Silva, Ian Victor
Ovarian cancer (OVCA) is the most lethal gynecological cancer, and its dissemination and chemoresistance are major factors determining disease prognosis. Better understanding of mechanisms involved in these processes is crucial to improve OVCA therapy; therefore being the aim of the present study. Firstly, the role of NAC1 and BCL6 regulating transcription in OVCA has been investigated using FOXQ1 as a study model. It has been shown that NAC1 and BCL6 interact through NAC1’s BEN C-terminus domain, forming a complex which binds to three BCL6 binding motives on FOXQ1 promoter, activating its transcription. A positive correlation between NAC1 and BCL6 expression has been shown in OVCA cell lines and tumor specimens. Moreover, BCL6-depending NAC1 binding to BCL6 promoter activating its transcription has been shown. Therefore, a novel mechanism by which a BTB/POZ family member interacts with BCL6 attenuating its auto-repression has been herein described. Finally, cDNA microarray analyzes revealed a plethora of putative NAC1/BCL6 target genes. On the second chapter, the effects of cisplatin on OVCA progression have been analyzed. It has been shown that a five-day treatment with 10-5M cisplatin of A2780 cell increased chemoresistance, cell migration and expression of the cancer-stem cell associated phenotype CD44+CD24-, and a G2/M cell cycle arrest. The secretion of TGF-β1 and CXCL2, both cytokines involved in migration and resistance in cancer, in response to cisplatin, doxorubicin, and paclitaxel, by three cell lines originated from serous (A2780), endometrioid (MDAH-2774), and clear cell (TOV21G) OVCA subtypes has been measured. In response to cisplatin, TGF-β1 secretion by TOV21G cells has increased, as has CXCL2 secretion by TOV21G and A2780. When analyzing the effect of exogenous CXCL2 in drug resistance, this relation could not be demonstrated. However, it could be explained by CXCR2 overexpression in response to cisplatin, which has been herein demonstrated. Therefore, several different mechanisms leading to OVCA progression have been identified.
Estudo da expressão de PHD3, HIF1-α e SOD1 em modelo experimental de câncer colorretal tabagista e resposta a radioterapia
(Universidade Federal do Espírito Santo, 2016-05-19) Trivilin, Leonardo Oliveira; Conforti, Adriana Madeira Álvares da Silva; Paula, Flávia de; Nogueira, Breno Valentim; Silva, Eloiza Helena Tajara da; Boeloni, Jankerle Neves
Smoking is a risk factor for colorectal cancer development and interferes on protein expression involved on tumoral progression, prognostic and therapeutic response. Thus, the objective was to study PHD3, HIF1-α and SOD-1 expression, as well as response to radiotherapy in experimental model for colorectal cancer exposed to cigarette smoke. It was used 53 young male rats Wistar, weighing on average 181,35g (±18,7g). Five animals were kept as negative control DMH-/tobacco- (G0).The induction of colorectal carcinogenesis with DMH during five weeks has been conducted on 48 animals, divided on experimental groups consisting of 12 animals each: DMH+ group (G1), DMH+/radiotherapy group (G2), DMH+/tobacco+ group (G3) and DMH+/tobacco+/radiotherapy group (G4). The exposure of G3 and G4 groups to cigarette smoke occurred in inhalation chamber equipped with smoke puff, and corresponded to 12 cigarettes/day divided into two exposure shifts of 60 minutes each, over 143 days. In the 21th experiment week, G2 and G4 animals underwent three radiotherapy sessions at a dose of 700 cGy each, totaling 2500 cGy. At 22 weeks, all animals were euthanized for removal of >0.1 cm injuries, processed histologically and stained with Hematoxylin-Eosin for diagnosis. It was selected samples classified as tubular adenocarcinoma (G1, G2, G3, G4) and normal mucosa for immunohistochemical technique for proteins PHD3, HIF1-a, SOD1 and cleaved caspase-3. The radiation response was obtained by apoptotic index of G2 and G4 groups. In G1 group were found dysplastic lesions, benign and malignant, moderate to severe inflammation and severe to moderate pleomorphism. In G3 were found dysplastic and malignant lesions, mild to moderate inflammation and mild to moderate pleomorphism. PHD3 protein suffered downregulation in G1, G3 and G4, more expressed in G2 (p=0.0011). HIF1-α protein was less expressed in G1 (p=0.0005) and increased its expression in this group after radiotherapy compared to others. SOD-1 was more expressed in G1, and after radiotherapy in G4 (p=0.005). The apoptotic index was significantly higher in G4 (p = 0.0289). Malignancies were predominant in exposed to cigarette smoke experimental model indicating its use in studies of smokers prognostic and survival, through use of molecular tools and, PHD3 downregulation, HIF-1alpha upregulation and levels of SOD-1 lower than unexposed animals, led to better radiotherapy response.
Proteômica quantitativa, livre de marcação, de Carica papaya L. em resposta à doença da meleira do mamoeiro
(Universidade Federal do Espírito Santo, 2016-06-29) Soares, Eduardo de Almeida; Silas, Pessini Rodrigues; Fernandes, Patricia Machado Bueno; Ventura, José Aires; Silva, Diolina Moura; Romão, Wanderson; Zingali, Russolina Benedeta
Papaya (C. papaya L), a fruit of great economic importance worldwide, which has suffered huge preharvest losses, mainly by papaya sticky disease (PSD), characterized by spontaneous exudation of aqueous and fluid latex, which oxidizes and accumulates as a sticky substance in the organs of the plant. PSD is caused by a synergic infection by PMeV and PMeV2 viruses, whose symptoms arise only after the juvenile-adult transition (flowering) of the plants. To understand the plant-virus interaction mechanisms and the phenological dependence of the symptoms onset, the C. papaya proteome was accessed by LC-MS/MS-based label-free quantitative proteomic approach for infected and uninfected (control) plants in four different ages (3, 4, 7 and 9 months post germination). This study permitted the identification of 1,623 and quantification of 1,609 proteins, whose the abundances comparison showed an increased levels of photosynthesis related proteins and decreased levels of proteins related to caspase-like activity, 26S-proteasome and cell wall remodeling during asymptomatic stage (prior to the flowering). The onset of the symptoms after flowering (7 months after germination) was accompanied by a reduction in the accumulation of proteins related to photosynthesis and increase in accumulation of proteins related to the metabolism of carbohydrates, lipids, amino acids, proteins, nucleotides and nucleic acids. In addition, was observed the accumulation of proteins involved in response to stress, signaling, transport and cell wall. The sum of these results supports the hypothesis of an incomplete tolerance mechanism in the asymptomatic phase (prior to flowering), with a chloroplast ROS signaling followed by ineffectiveness in containing systemic infection by activity depletion of caspase-like, proteasome, and cell wall remodeling. This incomplete tolerance mechanism at pre flowering acquire new elements with the juvenile-adult transition, which the installed systemic infection, delivers the late and ineffective symptoms of necrotic and chlorotic response. Inhibition in cell wall remodeling processes prior to flowering weakens the latex vessels, which bursts during the PSD osmotic imbalance, leading the sticky aspect of the diseased papaya plants.
Biomarcadores da longevidade humana e bioatividade do exopolissacarídeo botriosferana no envelhecimento
(Universidade Federal do Espírito Santo, 2016-07-15) Sena, Geralda Gillian Silva; Batitucci , Maria do Carmo Pimentel; Paula, Flavia de; Zeidler, Sandra Ventorin von; Marin-Morales, Maria Aparecida; Errera, Flávia Imbroisi Valle; Morelato, Renato Lírio
In the last decades it has been observed a world grow in elderly population associated with the increase of longevity. Multiple factors, among them, environmental, behavioral and genetic can influence human longevity. There is a great interest in improvement of natural products with functional and/or health proprieties. Fungal exopolysaccharides (EPS) as (1→3;1→6)-βD-glucan botryosphaeran, secreted by Botryosphaeria rhodina MAMB-05, are promising candidates for being considered modifiers of biological response. The present study aimed: a) to investigate in human possible longevity biomarkers through the frequency of polymorphisms at the genes FOXO3 (rs2802292), SOD2 (rs4880), APOE (rs429358 and rs7412) and SIRT1 (rs2273773) in a sample of elders of Grande Vitória, ES, as well as its state of oxidative stress and DNA integrity level; b) asses antimutagenic, mutagenic and cytotoxic of botryosphaeran in young and aged Swiss mice, from both gender, as well as its hypolipidemic, hypoglycemic and antiatherogenic potential in older male LDL receptor knockout (LDLr-/-) animals and its background (C57BL/6). To achieve the objectives: a) in elderly sample, it was characterized demographic, socioeconomic, anthropometrics, biochemical, clinics and life style data. Genetic polymorphisms were analyzed through real time polymerase chain reaction; the malondialdehyde, by high performance liquid chromatography and genomic damage, by alkaline comet assay in groups of long-lived individuals and controls (≥ 85 years and 70-75 years); b) with animals - botryosphaeran, was administrated, by gavage (doses of 7.5, 15 and 30 mg/kg b.w. per day) in a 30-day pretreatment in 30-day protocol (young mice) and 15-day protocol (older mice) to investigate its mutagenic and anticytogenotoxic potential against damages induced by cyclophosphamide. The micronucleus assay was carried through erythrocytes of peripheral blood and bone marrow from mice. Glucolipidemic and atheroprotective effects of EPS (30 mg/kg b.w. per day, by gavage) among LDLr-/- animals, that received atherogenic diet, were verified by plasmatic glucose measure and lipidic profile after 15 days of treatment, with commercial colorimetric kits. The atherosclerotic lesion was quantified by aortic lipidic deposition analysis (en face), with Oil-Red-O. The statistical analysis was performed by χ² test, Fisher exact test, Tukey test, t Student, Mann-Whitney and Hardy-Weinberg Equilibrium (H-WE) (p<0.05). Among oldest-old individuals and controls, the plasmatic levels of malondialdehyde and DNA damage had similar values. It was observed a positive association between rs2802292 FOXO3 and longevity. Biochemical and anthropometric characteristics, related to successful aging, showed significant results. In in vivo assay, botryosphaeran, in the 3 doses, 14 it was not mutagenic and still reduced the percentage of damage between young and older animals (Swiss, C57BL/6 e LDLr-/-). There was reduction in glucose plasmatic levels (36%), improved in lipidic profile (reductions of 53.8-84.3%) and decreased of aortic lipidic deposition (32.8%) in the LDLr-/- atherosclerotic mice treated with EPS. Our results provide new insights of human longevity, in Brazilian population, and contribute to a promising future of genomic geriatric and personalized medicine. Moreover, it does indicate that botryosphaeran has relevant biologic effect, making it a promising candidate for new therapeutic products development.
Bioatividade de fitocompostos da Aloe vera barbadensis em modelos de psoríase In Vitro e In Vivo
(Universidade Federal do Espírito Santo, 2016-07-21) Amorim, Gustavo Modesto de; Sze, Daniel Man-Yuen; Rangel, Leticia Batista Azevedo; Piva, Terrence J.; Errera, Flavia Imbroisi Valle; Guimarães, Marco Cesar Cunegundes; Kuster, Ricardo Machado; Gouvea, Sonia Alves
Psoriasis is an autoimmune, chronic inflammatory disease, affecting 2-3% of the world population. In the early stages of the disease onset, studies have shown chemerin as a triggering protein playing a key role. Brazilian national essential medicines list (RENAME) proposed the topic usage of Aloe vera barbadensis extract as a therapeutic approach for psoriasis for its immunomodulatory and anti-inflammatory effects. In order to investigate them, we adopted an In Vivo murine model with phenotype similar to psoriasis via topical application of imiquimod (IMQ) and treating animals with 70% Aloe vera cream for further comparison with controls. In addition, animal skin biopsies were tested for the enzymatic activity of the myeloperoxidase (MPO) as an indicator of neutrophil activity. As In Vitro model it was used an immortalized keratinocyte cell line to assess the wound healing potential of the Aloe vera freeze dried extract 200:1 (AVFDE) via Scratch Assay. Later, primary human dermal fibroblasts were used to evaluate the potential modulation on constitutive expression of chemerin and three proinflammatory cytokines. Culture supernatants of dermal fibroblasts were assessed against AVFDE and five of its phytoconstituents including the polyssacharide Acemannan (ACM). For this, we used ELISA and flow cytometry. We partially reproduced In Vivo human psoriatic phenotype in mice via IMQ induction and the MPO activity was shown to be lower in animals treated topically with 70% Aloe vera cream. It was observed in the In Vitro experiments that AVFDE 50ug/ml reduced the expression of MCP-1 chemokine and the concentration of 250ug/ml increased IL-8 expression. ACM 250ug/ml increased the expression levels of IL-8 and IL-6. Acemannan (ACM) at the two tested concentrations significantly reduced chemerin expression. Aloeresin A 20uM increased IL-6 expression. Either tested phytosterol, at any concentration range elicited effects on the expression levels of cytokines or chemerin. The results suggested that the therapeutic usage of Aloe vera extract as an immunomodulatory and / or anti inflammatory agent must be further evaluated. Although the extract demonstrated a potential benefit in the treatment of early stages of psoriasis it is essential that commercial plant extracts are standardized with a minimum content of bioactive phytocompounds as Acemannan within the marketed pharmaceutical formulations as stated by related regulatory organisms.
Poliformismos nos genes TCF7L2 e ADIPOQ e sua associação com obesidade e diabetes em adolescentes da Grande Vitória
(Universidade Federal do Espírito Santo, 2016-08-30) Costa, Josivany Valério de Freitas; Errera, Flavia Imbroisi Valle; Paula, Flavia de; Maranduba, Carlos Magno da Costa; Bem, Daniela Amorim Melgaço Guimarães do; Batitucci, Maria do Carmo Pimentel; Zeidler, Sandra Ventorin von
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