Mestrado em Ciências Biológicas (ICB)

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Ação antimicrobiana de enzimas hidrolíticas produzidas por Trichoderma asperellum e imobilizadas em blendas de polímeros biodegradáveis.
(Universidade Federal de Goiás, 2011-01-28) SILVA, Barbara Dumas Santos; ULHOA, Cirano Jose; http://lattes.cnpq.br/8368469162867277; FERNANDES, Kátia Flávia; http://lattes.cnpq.br/9737543228759171
The hydrolytic activity of enzymes produced by Trichoderma asperellum, immobilized biodegradable films, as growth inhibitor of microorganisms was tested. The inhibitory activity was demonstrated on Aspergillus niger, Penicillium sp. and Sclerotinia sclerotiorum, microorganisms usually related to the attack and/or food contamination at the field or packaged. We used two polymer blends with different compositions, cassava starch and poly-butylene adipate-co-terephthalate (Ecoflex®, BASF Chemical Company) and other composed for polyvinyl alcohol (PVA) and polysaccharide cashew gum (PEJU). T. asperellum was induced to produce enzymes involved in the attack mycoparasite (N-acetylglucosaminidases, β-1,3-glucanases, chitinases and proteases) by the addition of crude chitin in the growth medium. The enzymes produced in major quantity were N-acetylglucosaminidase and chitinase. The pool of enzymes produced in the experiments was then used for immobilization tests. The immobilization process was performed in films by two methods: covalent and ionic bonding. In both methods, the presence of immobilized hydrolytic enzymes resulted in reduced growth of microorganisms, but the covalent immobilization of the results were more expressive. S.sclerotiorum was the microorganism most sensitive, followed by A. niger and Penicillium sp. To confirm the action of hydrolytic enzymes produced by T. asperellum and evaluate the effects they produce cell wall of microorganisms and other structures, the films with enzyme immobilized by covalent bonding were subjected to scanning electron microscopy. The structures most affected were hyphae and spores. Overall, the synergistic action of all enzymes produced by T. asperellum, reduced the growth of microorganisms when immobilized on the surface of the films Starch-Ecoflex® and PVA-PEJU. Moreover, the polymer blends tested exhibited desirable characteristics for future use in food packaging and most importantly, also provide efficient systems for the immobilization of enzymes.
Ação da fosfolipase B extracelular de Paracoccidioides brasiliensis na interação ex vivo com macrófagos alveolares
(Universidade Federal de Goiás, 2010-03-26) SOARES, Deyze Alencar; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the most prevalent systemic mycosis in Latin America, paracoccidioidomycosis. The phospholipase B (PLB) enzyme is considered an important virulence factor in this dimorphic fungus, involved in the immune response of the host-pathogen interaction. Our objective was to determine whether a P. brasiliensis (Pb18) PLB is involved in adhesion / internalization of yeast and evasion of host immune responses. The effect of PLB was analysed using specific inhibition of PLB (alexidine dihydrochloride) and pulmonary surfactant in an ex vivo model (Pb18) of alveolar macrophage (MHS cells) infection. PLB enzyme assays and real time RT-PCR (qRTPCR) analysis of genes differentially expressed in the process of evasion: plb1 (phospholipase B1), icl1 (isocitrate lyase) and sod3 (Cu, Zn dismutase) and immune responses: clec2 (C-type lectin domain 2), cd14 (cluster of differentiation 14), tlr2 (toll-like receptor 2), nfkb (nuclear factor kappa B), nkrf (NF-kappaB repressing factor), il1β (inteleukin-1β) and tnfα (tumor necrosis factor alpha) were carried out using selective inhibition of PLB activity and pulmonary surfactant. The levels of cytokines inteleukin 10 (IL-10), IL-12 and TNF-α) were also determined by ELISA. PLB activity under adhesion conditions of P. brasiliensis (Pb18) to alveolar macrophage cells was found at high levels up to 6 hours post-infection. In the conditions of exposure to pulmonary surfactant and alexidine dihydrochloride, PLB activity and the level of transcripts of genes related to phagocytosis and inflammatory response were measured. We found that PLB activity had an influence on the phagocytic activity of alveolar macrophages. Alexidine dihydrochloride (0,25 μM) selectively inhibited PLB activity by 66% and decreased significantly the adhesion and internalization of yeast on MHS cells. Genes involved in phagocytosis (trl2 and cd14) and inflammatory response (nrkf, tnfα and il1β) were down-regulated in the presence of the PLB inhibitor. In contrast, the PLB activity and internalization of fungal yeast cells increased significantly in the presence of pulmonary surfactant (100 μg/mL) and genes such as clec2, important for effective phagocytosis by MHS cells, and the pro-inflammatory inhibitor (nkrf) were up-regulated. Also, the pulmonary surfactant did not alter cytokine production, while alexidine dihydrochloride decreased the levels of IL-10 and increased the levels of IL-12 and TNF-α. In addition, through simultaneous analyses of gene expression for the pathogen, P. brasiliensis, we found upregulation of the genes sod3, icl1 and plb1, required for the evasion of alveolar macrophages. P. brasiliensis PLB is important for the binding and internalization of yeast at macrophage surfaces. The specific effect of inhibiting PLB enzyme activity indicates that adhesion may be facilitated indirectly via fatty acid release from phospholipids of the membrane of host cells. This is the first study to show that PLB activity may modulate immune responses to P. brasiliensis infection.
Alterações cardiovasculares induzidas pela administração aguda e crônica do protótipo de fármaco LQFM-21
(Universidade Federal de Goiás, 2015-02-26) Trindade, Neidiane Rosa; Menegatti, Ricardo; Pedrino, Gustavo Rodrigues; http://lattes.cnpq.br/1155446449250341
This study aimed to investigate the effects hypotensive (LQFM-21), trying to identify a pharmaceutical potential for this new drug prototype. Male Wistar spontaneous hypertensive rats (SHR) and wistar rats were used to evaluate the effect of LQFM-21 on mean arterial pressure (MAP), heart rate (HR), renal vascular conductance (RVC), arterial vascular conductance (CVA) and baroreflex sensitivity index (BI). The administration of LQFM-021 (0.05, 0.1, 0.2, 0.4 mg / kg, iv.) Decreased MAP (-9.0 ± 1.4, -21.5 ± 2.9, ± 2.8 - 26.7 mmHg respectively); increased RVC (12.0 ± 1.9, 23.2 ± 3.2, 26.8 ± 6.3, 34.8 ± 5.9%, respectively) and CVA (17.3 ± 1.7; 27 8 ± 2.6, 32.7 ± 4.8 and 42.2 ± 3.4%, respectively) in anesthetized normotensive rats. In SHR, also found reduction of MAP (-16.9 ± 2.8; -19.4 ± 2.5; -22.0 ± 3.8 -17.2 ± 6.4 mmHg, respectively), HR (-11.2 ± 0.8 bpm at a dose of 0.04 mg / kg) and an increase in RVC (22.2% at a dose of 0.2 mg / kg) and CVA (12.2 (12.2 ± 2.1, 13.8 ± 5.6, 18.2 ± 2.3, 15.6 ± 3.1% at doses of 0.05, 0.1, 0.2, 0.4 mg / kg, respectively). Both the acute, oral as the oral administration chronicle of LQFM-21 at a dose of 15 mg / kg reduced the MAP in animals wistar 89.9 ± 1.9 mmHg and 136.4 ± 4.9 mmHg SHR without changing BI in wistar animals with the infusion of phenylephrine -2.3 ± 0.32 vs 1.9 ± 0.1 mmHg / bpm and SHR -1.9 ± 0.2 vs. -1.9 ± 0.3 mmHg / bpm, with infusion nitroprusside in wistar 3.2 ± 0.31 vs 3.0 ± 0.04 bpm / mmHg and 3.9 ± 0.6 vs SHR 3.0 ± 0.04 bpm / mmHg. Together our results demonstrated hypotensive effects of LQFM- 21. However further studies are needed to identify the mechanism of action and toxicity of this drug prototype.
Alterações cardiovasculares induzidas por supernutrição durante a lactação em ratos Wistar na idade adulta
(Universidade Federal de Goiás, 2019-02-18) Ferreira Junior, Marcos Divino; Castro, Carlos Henrique de; http://lattes.cnpq.br/6354834854727314; Gomes, Rodrigo Mello; http://lattes.cnpq.br/3121095341590269; Gomes, Rodrigo Mello; Mendes, Elizabeth Pereira; Matafome, Paulo Nuno Centeio
Metabolic syndrome, closely related to obesity and its comorbidities, has become a worldwide pandemic, with increasing projections each year. The role of adipose tissue in the metabolic syndrome is not limited to the accumulation of lipids but also to the secretion of adipokines, which can control food consumption and energy expenditure. In obese individuals, there is a change in adipokines profile, from anti-inflammatory to pro-inflammatory. Inflammatory status in adipose tissue leads to insulin resistance, increased adipocyte differentiation and macrophages recruitment. In addition, the angiotensinogen (AGT) produced by adipose tissue may correspond to more than 30% of plasma levels of this substance, which increases in case of adipocyte hypertrophy. AGT is a key part of the Renin Angiotensin System (RAS), and is directly linked to circulating Angiotensin II (ANG II) levels, and the latter is linked to obesity-induced cardiovascular changes. Several studies have proposed to evaluate the effects of obesity on cardiovascular parameters, but the majority in animals with diet-induced obesity. Here, we aimed to evaluate the effects of postnatal overnutrition-induced obesity (PNO), due to litter reduction (SL), in metabolic and cardiovascular parameters in adulthood. In our study, SL group developed hyperphagia, greater adiposity and body weight, and dyslipidemia. Also, important cardiovascular alterations were developed in SL animals, such as hypertension, left ventricular hypertrophy, endothelial dysfunction, and increase of oxidative stress related proteins. However, through in vivo and ex vivo cardiac function analyzes, our results suggest that the hearts of animals in the SL group present neurohumoral compensation of their function, which leads to overload and may progress to heart failure. Taken together, our data may help in the elucidation of mechanisms by which obesity-induced cardiovascular changes occur, increasing the possibility of discovering new targets for treatment.
Alterações dos parâmetros de comportamento de ratos tratados com peptídeo rico em prolina da serpente Bothrops jararaca, Bj-PRO-7a
(Universidade Federal de Goiás, 2018-05-16) Turones, Larissa Córdova; Ianzer, Danielle Alves; http://lattes.cnpq.br/7609262674053858; Custódio, Carlos Henrique Xavier; http://lattes.cnpq.br/0207928273284808; Pansani, Aline; Gomes, Rodrigo Mello
The Bj-PRO-7a, a heptapeptide isolated and identified from Bothrops jararaca (Bj) venom, evoke potent therapeutic effects, as antihypertensive effect, natriuretic and diuretic effects, promotion of angiogenisis and vasodilatation. The effects of heptapeptide are independent of Angiotensin Converting Inhibitor (ACE), possibly dependent of Muscarinic Receptors subtype 1 (M1R) activation and oxido nitric pathways. Also, the Bj-PRO-7a actions upon central nervous system still need to be evaluated. Thus, the aims of this study were: i) to assess the effects of acute administration of Bj-PRO-7a upon behavior; ii) to reveal mechanisms involved in the effects of Bj- PRO-7a upon locomotion/exploration, anxiety and depression-like behaviors. For this purpose, adult male Wistar (WT) and Spontaneous Hypertensive Rats (SHRs) (300-380 g) received i.p. injections of Vehicle (NaCl 0.9%), Diazepam (2 mg/kg), Imipramine (15 mg/kg), Bj-PRO-7a (71, 213 or 426 nmol/kg), Pirenzepine (852 nmol/kg), α-metil-DL-tirosina (200 mg/kg) or Chlorpromazine (2 mg/kg) and were placed in the Elevated Plus Maze (EPM), Open Field (OF) and Forced Swimming (FS) tests. The heptapeptide promoted anxiolytic and antidepressantlike effects and increased the locomotion/exploration. These effects of Bj-PRO-7a seem to be strongly dependent on activation of M1R, catecholaminergic paths and dopaminergic receptors.
Análise do secretoma de isolados do fungo Trichoderma asperellum (TR356) em resposta ao fungo fitopatogênico Sclerotinia sclerotiorum
(Universidade Federal de Goiás, 2014-10-23) Rodrigues, Amanda Rafaela; Ulhoa, Cirano José; http://lattes.cnpq.br/8368469162867277; Ulhoa, Cirano José; Monteiro, Valdirene Neves; Fernandes, Kátia Flavia
The genus Trichoderma is represented by non-pathogenic soil fungi that have been studied by act as biological control agents against fungal pathogens, such as Sclerotinia sclerotiorum, the fungus that causes white mold, besides that, reduce environment and human health risks by being able to replace agrochemicals. Proteomic strategies with MS techniques have been important tools in studies of patterns identification for protein expression in different growth conditions. This work aims to development new strategies that enable the detection and identification of proteins secreted by Trichoderma asperellum (TR356) and Sclerotinia sclerotiorum when inoculated together and separated. The results obtained by MALDI / TOF analysis allowed the identification of a β-1.3--glucanosiltransferase and α-1.2-Dmannosidase, demonstrating the possibility of proteins identification for better comprehension about interaction between these organisms. It was therefore possible to identify the proteins through strategies used, but further analysis are required in order to elucidate the function and interaction of proteins secreted by the fungus Trichoderma asperellum TR356 against S. sclerotiorum.
Análise da diversidade genética em populações de Sclerotinia sclerotiorum
(Universidade Federal de Goiás, 2010-08-31) Nascimento, Lucas Breseghelo do; Silva, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373
The fungus Sclerotinia sclerotiorum is among the most successful pathogens, omnivores, and without specific hosts, is considered one of the most important fungal pathogens in the world. Is distributed in all producing regions, temperate, subtropical or tropical. The fungus produces resistant structures called sclerotia on the surface and within tissues colonized, they returned to the soil with crop residues and are responsible for the survival of the fungus in the same area for up to eight years. The objectives of this study were to quantify the variability within and between populations of the fungus S. sclerotiorum in bean and soybean crops in different producing places of those varieties. 46 isolates were collected of S. sclerotiorum in six locals of grain production in different regions of Brazil. The sites chosen were Monte Carmelo-MG, Formosa-GO, Lucas do Rio Verde-MT, Montividiu-GO, Londrina, PR and Santo Antonio de Goiás-GO. All areas of the original host of the gathering was the bean with the exception of Londrina-PR in which the host was soybeans. A population study of the fungus through RAPD markers using 13 primers was carried out for the analysis of genetic variability of the fungus. In parallel, tests were also made for the the Mycelial compatibility groups among isolates and test production of hydrolytic enzymes. The statistical analysis was performed using the Arlequin software, which indicated variability among populations of 16.94% and 83.06% within populations. Were found 10 mycelial compatibility groups without specific populations .Enzyme activities performed indicated significant differences within the populations of all enzymes, a comparison between populations also showed significant differences among populations in polygalacturonases, 1,3-β-glucanase and xylanase. The results indicate a high level of variability within populations and low variability among populations.
Análise da expressão de genes envolvidos na manutenção da homeostase de cobre no patógeno humano Paracoccidioides brasiliensis
(Universidade Federal de Goiás, 2009-05-29) SANTOS, Rodrigo da Silva; SOARES, Célia Maria de Almeida; http://lattes.cnpq.br/8539946335852637
The fungus Paracoccidioides brasiliensis is a human pathogen with a wide distribution in Latin America. The fungus causes paracoccidioidomycosis when mycelia reachs the lungs. The success of the infection depends on the acquisition of essential micronutrients such as copper, which is required as cofactor for a variety of enzymes important in biological in several processes, such as respiration, growth and acquisition of iron. Previous studies, of the Laboratory of Molecular Biology showed that a high affinity copper transporter (PbCTR3) is a molecule highly expressed and probably necessary for the infection establishment of by P. brasiliensis. In the present study were isolated and characterized the genomic and cDNA sequences encoding for PbCTR3 of P. brasiliensis. The cDNA presented 582 base pairs and encodes for a protein with 193 amino acids, predicted molecular mass of 21.5 kDa and pI of 8.6. The genomic sequence has four exons interrupted by three introns. In silico analysis was performed on the database of the structural genome of P. brasiliensis (http://www.broad.mit.edu/annotation/genome/paracoccidioides_brasiliensisMultiHome.html) , where genes involved in maintaining the homeostasis of copper have been identified and used to design of a model of copper homeostasis in P. brasiliensis. The transcriptional behavior of Pbctr3 and genes involved in copper homeostasis were examined during exposure of yeast cells of P. brasiliensis to copper and iron depletion conditions, by real time qRTPCR. It was demonstrated a significant change in transcription lovel those of genes in the absence of copper, as well as in the combined absence of both metals. qRT-PCR was used to analyze the expression of Pbctr3 and Pbcrp (which encodes a protein responsive to copper) in yeast cells of P. brasiliensis derived from infected lungs and spleen at different time points of infection. The expression of Pbctr3 and Pbcrp was super-regulated during experimental infection. Taken together, these data suggest the importance of PbCtr3 and of absorbing copper / iron during the infectious process
Análise da influência dos polimorfismos 1236C>T, 2677G>T/A E 3435C>T do gene ABCB1 na resposta ao tratamento com clozapina
(Universidade Federal de Goiás, 2015-12-04) Salamanca, Ayda Luz Malaver; Santos, Rodrigo da Silva; Ghedini, Paulo César; http://lattes.cnpq.br/5789550234984454; Ghedini, Paulo César; Brito, Rodrigo Bernini; Bicudo, Lucilene Arilho Ribeiro; Santos, Rodrigo da Silva
Clozapine (CLZ) is the antipsychotic drug of choice in treatment refractory schizophrenia (TRS), however 30% of the patients with TRS don’t have full response to treatment with CLZ, these patients are considered to have super refractory schizophrenia (SRS). The response variability to treatment with CLZ may be associated with alterations of the CLZ plasma levels promoted by 1236C>T, 2677G>T/A e 3435C>T ABCB1 gene polymorphisms. ABCB1 is a transmembrane glycoprotein of transport, which export drugs from the intracellular to the extracellular space. ABCB1 polymorphisms cause structurally and functional protein changes that influence the intracellular CLZ levels and, consequently, promoting therapy failure. This work had the aim to establish the relation between the 1236C>T, 2677G>T/A e 3435C>T ABCB1 polymorphisms and the CLZ treatment response. This study included a total of 68 patients, 34 of whom were classified as TRS (CLZ responders) and 34 as SRS (CLZ non-responders). All patients were in use of CLZ for at least one year. For the genotype test was extracted genomic DNA, following for PCR and sequencing techniques. It was not observed any differences in allelic and genotype distribution between 1236C>T, 2677G>T/A and 3435C>T polymorphisms between RS and SRS groups. The polymorphisms not influenced the CLZ dose level, and percentage of BPRS change. In the same way, the results not showed relation between age, coffee intake, smoking behaviors and ethnicity and the CLZ treatment response, however, was evidenced a higher proportion of female patients in the SRS group when compared with the TRS group. In opposite, a lower proportion of male patients was observed in the SRS than the TRS group. Taken together, the results here obtained showed no association between 12361236C>T, 2677G>T/A e 3435C>T polymorphisms of ABCB1 gene and failure to CLZ treatment.
Análise da tolerância á resistência em Trichoderma harzianum
(Universidade Federal de Goiás, 2017-07-31) Santos, Karina Roterdanny Araújo dos; Georg, Raphaela de Castro; http://lattes.cnpq.br/3971276154067590; Georg, Raphaela de Castro; http://lattes.cnpq.br/3971276154067590; Campos, Ivan Torres Nicolau de; Izacc, Silvia Maria Salem; Monteiro, Valdirene Neves; Lorenzón, Esteban Nicolás
The presence of heavy metals in the soil has increased, causing many problems of an agricultural nature. Trichoderma are a species which is among the most well know and used biocontrol agents wordwide, as well as possessing qualities such tha parasitism and antibiosis, they have great resistance to metals and can also absorb them. Therefore, it is necessary for more detailed studies of the resistance of this fungus to metals and to research further the processes involved. In this work, we aimed to better understand the tolerance of T. harzianum to aluminum chloride, though a series of experiments performed in BDA, MEX e MYG, as well Bradford tests for quantification of proteins. In addition, we evaluated the gene response to metal stress through the sequencing RNA samples obtained when the fungus was created in BDA medium and submitted to concentrations of 1.5 and 3.0 mg / mL of aluminum chloride, 1.5 mg / mL, the fungus showed significant inhibition. It was observed that T. harzianum showed higher mycelial growth when grown in BDA medium compared to MEX and MYG, presenting levels of protein secretion inversely proportional to increasing concentrations of aluminum. The amount of transcriptionspecific factors of the stress response was increased, as well as the induction of genes involved in G-mediated cell signaling and increase in genes involved in vacuolar transport and carrier proteins. A repression of genes encoding proteins associated with cellular processes important for the growth of T. harzianum was also observed. These results show that T. harzianum is resistant to aluminum since it is able to tolerate and absorb large concentrations of this metal, which is commonly harmful to agriculture.
Análise das proteínas de reserva do arroz silvestre oryza glumaepatula e de linhagens interespecíficas oryza sativa x o. Glumaepatula
(Universidade Federal de Goiás, 2010-03-31) SANTOS, Karina Freire D'eça Nogueira; DIDONET, Claudia Cristina Garcia Martin; http://lattes.cnpq.br/2890489628230874; BRONDANI, Claudio; http://lattes.cnpq.br/4775600104554147
The objective of this dissertation was to analyze quantitative and qualitatively the total storage protein content and its fractions albumin, globulin, prolamin and glutelin from the grain of 29 genotypes of the wild rice Oryza glumaepatula and 70 interspecific lines backcross 2 obtained from the cross O. sativa x O. glumaepatula. From these lines, 34 BC2F8 were obtained from the cross O. sativa BG 90-2 x O. glumaepatula RS-16 and 36 BC2F10 were obtained from the cross O. sativa CICA-8 x O. glumaepatula RS-16. From the analysis of variance for the total protein content and its fractions, it were found highly significant differences (P<0.01) between the wild genotypes and the interspecific lines. The average of total protein content of wild genotypes was 12.17%, whereas the lines of BG 90-2 x RS-16 showed an average of 7.05% and the lines of CICA-8 x RS-16 showed an average of 8.40%. The wild genotype BGA14280 showed the highest total protein content (14.94%). In the comparative analysis of interspecific lines and their parents, it were found five lines with higher total protein content (average of 10.95%), which was significantly superior to the cultivated parent BG 90-2 (10.0%) and CICA-8 (9.61%). However, these lines showed lower content in relation to the wild parent RS-16 (14.06%). In relation to the protein fractions, 40 interspecific lines showed higher values in relation to their parents, excepting in one occasion, where the wild parent RS-16 showed higher value to the glutelin fraction. Considering the 29 wild genotypes, it were found the highest contents of albumin, prolamin and glutelin, excepting to the globulin fraction, where the wild genotype was not significantly different from the cultivar BRS Bonança. The SDS-PAGE analyses of the total protein and the glutelin fraction showed a differential profile of α-glutelins for the wild genotypes, emphasizing the BGA14232 genotype, which did not showed the α-polypeptides commonly identified in the remaining genotypes. Considering the interspecific lines and their parents, it was found similar profile of total protein and α-globulins, with differences in relation to the 40 kDa α-glutelin, which was found just in the wild parent RS-16, indicating a differential expression in O. glumaepatula, since the O. sativa showed a 39 kDa α-glutelin. In relation to albumin, globulin and prolamin fractions, the wild genotypes showed a different protein profile in comparison to the interspecific lines and the cultivated rice, probably due to the two crosses in direction to BG 90-2 and CICA-8, which were the recurrent parents during the interspecific lines development. The highest protein content found to the O. glumaepatula, the different protein profile and the finding of interspecific lines with higher protein content in relation to their cultivated parents clearly show the positive contribution of this species to the genetic breeding aiming the increase of the nutritional value for the grain of the cultivated rice in Brazil.
Ánálise de alterações no gene receptor de andrógeno em homens com infertilidade idiopática
(Universidade Federal de Goiás, 2009-03-31) MESQUITA, Wyara Elanne de Jesus Castro; JESUÍNO, Rosália Santos Amorim; http://lattes.cnpq.br/5113656623817587
Male idiopathic infertility is related to defects in normal spermatogenesis, due to genetic causes. The spermatogenesis is a dependent process on high levels of male sex hormones, the androgens. The androgen, in turn, perform its function when associated with the androgen receptor (AR), protein encoded by AR gene. Mutation in AR gene lead to a synthesis of non functional AR, which results in the failure of the process of spermatogenesis and, consequently, causes male infertility. This work has as its main objective the verification of the occurrence of mutation in the AR gene in patients with male idiopathic infertility who come from the HC-UFG Human Reproduction Center. Samples were analyzed from 206 patients. The result was that 95 patients were found to be normal while 111 with an altered result for the spermogram. The samples were amplified for exons 1, 4, 6, 7 and 8 of the AR gene and the results subjected to statistical analysis, Mann Whitney, logistic regression, and chi tests. The existence of the relationship between defects of sperm and AR gene mutation was verified. The analysis of the relationship between the spermogram and the AR gene mutation in five evaluated exons was significant only for exons 1 and 7. Patients with numerical unsettled spermogram had a higher frequency of mutations in exon 7, teratozoospermics in exon 1 and exon 7 in astenozoospermics patients. Exons 4, 6 and 8 showed no meaningful statistical relationship in reference to the alteration of the spermogram. Among results related to social custom, alcohol proved to be significant for mutation in the AR gene. This study has reaffirmed the relationship between the presence of mutation in AR genes as probable causes of defects in spermatogenesis. Consequently, male idiopathic infertility depends not only on the genetic factor, but also on the association between this factor and the environment where man inhabits
Análise de diferentes misturas enzimáticas do fungo Humicola grisea var. thermoidea na hidrólise da fração de hemicelulose de bagaço de cana-de-açúcar
(Universidade Federal de Goiás, 2011-08-31) Faria, Syd Pereira; Castro, Carlos Henrique de; http://lattes.cnpq.br/6354834854727314; Faria, Fabrícia Paula de; http://lattes.cnpq.br/3739169267521003; Faria, Fabrícia Paula de; Lopes, Francis Júlio Fagundes; Ulhoa, Cirano José
The biomass consists mainly of cellulose, hemicellulose and lignin. Cellulose is the most abundant polymer xylan and the main component of hemicellulose. Enzymatic hydrolysis is an important step in the bioconversion of cellulose and hemicellulose fraction of lignocellulosic residues. However, for the conversion of the hemicellulose, the substrate must undergo a pretreatment step which allows the refining of biomass at the microscopic level, separating the same into its main components (cellulose, hemicellulose and lignin) or loosening the fibers of its components, facilitating the accessibility of enzymes to hemicellulose chain. The conversion of cellulose and xylan to glucose and xylose may be performed by a group of enzymes produced by bacteria and fungi. The thermophilic fungus Humicola grisea var thermoidea produces an efficient complex of cellulolytic enzymes (endoglucanases, cellobiohydrolases and β-glucosidase) and xylanolítics (endoxylanases and β-xylosidase) with high thermal stability when cultivated in different lignocellulosic substrates. The aim of this study was to analyze the efficiency of enzymes produced by H.grisea in the hydrolysis of the hemicellulose fraction of sugarcane bagasse (BCA). BCA assays used in the hydrolysis was subjected to alkaline pretreatment (Treatment I, II and III) and steam explosion. BCA pretreated was subsequently hydrolyzed with different enzyme mixtures using culture supernatant of the fungus H. grisea supplemented with recombinant enzymes endoxylanase (HXYN2r), cellobiohydrolase (CBH1.2r) and β-xylosidase (XYNB2r). The results showed that the pre-treatment was most suitable to steam explosion which revealed a yield of about 78% xylan. The best enzyme formulation was a mixture of the supernatant of culture of H. grisea (SHG) grown in wheat bran (WB) and BCA supplemented with HXYN2r (950 U), CBH1.2r (950 U) and XYNB2r (1.17 U).
Análise de populações de Sclerotinia sclerotiorum em cultura de feijoeiro através de marcadores SSR.
(Universidade Federal de Goiás, 2009-09-30) GOMES, Eriston Vieira; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373
79 Sclerotinia sclerotiorum isolates were collected under central pivot irrigations sistems, from Brazilian fields, divided in 4 populations (S2, S3, A and M) and analyzed to determine the genetic variability among and between populations using molecular markers based on microsatelittes. 10 primers were used and the amplification products were separate in poliacrilamida gels and the bands were silver stained. A total of 102 different haplotypes were identified, and the amount of haplotypes varied from 6 to 18 for locus. The genotypic diversity ranged from 65% to 91%. Analyses based on genetic diversity and fixation indices which indicate the variability between populations was 28.79% (FST = 28793) and the variability among populations was 71.21%. The Jaccard similarity index indicated that the populations S2 and A is genetically closer. The population S3 presented a similarity index of 0.44 compared with the populations S2 and A. The population M, originated from several collection sites, was considered genetically more distant showing a index of 0.46 compared with the others populations . The high variability between and among populations can indicate that, besides the possible introduction of new genotypes in the analyzed fields, could be having clonal and sexual reproduction in isolated of S. sclerotiorum from Brazilian cerrado.
Análise de transcritoma e expressão gênica de feijoeiro comum (Phaseolus vulgaris L.) submetido ao estresse abiótico
(Universidade Federal de Goiás, 2016-03-08) Pereira, Wendell Jacinto; Vianello, Rosana Pereira; http://lattes.cnpq.br/4523999698824309; Lanna, Anna Cristina; Brondani, Claudio; Vianello, Rosana Pereira
The common bean (Phaseolus vulgaris L.) is an important foodstuff strongly associated with an issue if global food security. Among the factors that affect their agricultural production, abiotic stress episodes listed as high risk and impact, can restrict the area under cultivation and crop yields. The characterization of high-throughput differential gene expression involved in plant response to abiotic stresses enables profile the genes, and the metabolic pathways, involved in the mechanisms of tolerance to abiotic stresses. In this study, the expression of genes related to response to drought stress in leaf and root tissue of two contrasting common bean genotypes for stress tolerance was evaluated by RNA-Seq. A new Phaseolus vulgaris transcriptome was built featuring 1,668 new loci and 23,169 new isoforms. In all, 54,807 transcripts were identified, found being distributed in 28,640 genes. Through the edgeR package (Bioconductor), they determined by differential expression analysis of leaf and root 1,242 differentially expressed genes. Of these, 458 genes had different expression in tolerant genotype (191 up-regulates and 267 down-regulated) when compared to the sensitive genotype. Functional annotation revealed in tolerant genotype predominance of genes in the categories enriched oxidoreductase activity, oxidation-reduction process, regulation of gene expression, regulation of macromolecule metabolic process and dioxygenase activity. By qPCR, of the 15 DEGs identified via RNA-Seq and selected for characterization, 74.42% were validated for differential expression. Additionally, a total of 151,283 variants were identified, of which 135,167 are SNPs and 16,115 are indels. Through functional annotation, performed in SnpEff v.4.2, were identified 330,378 effects caused by variants. The data used in this study were deposited in the Sequence Read Archive (NCBI) and will be available. The results of this study contribute to the expansion of knowledge about the gene mechanisms, as well as their functional variants, related to abiotic stresses tolerance in beans.
Análise do perfil de expressão de genes da família Hsp70 de Trichoderma asperellum (TR356) durante o micoparasitismo e estresses abióticos
(Universidade Federal de Goiás, 2018-02-16) Rodrigues, Thuana Marcolino Mota; Georg, Raphaela de Castro; http://lattes.cnpq.br/3971276154067590; Georg, Raphaela de Castro; Izaac, Silvia Maria Salem; Faria, Fabrícia de Paula
The genus Trichoderma ssp, is today the group of species most commonly studied and used to act in agriculture as biological control against phytopathogens. Its rapid mycelial growth, associated with high production of conidia, synthesis of several antibiotics and ability to live in different forms (saprotrophic, symbiont or mycoparasite) are characteristics that make it attractive for this purpose. The relation of mycoparasitism, as well as oscillations in environmental conditions, naturally causes cell stress and consequent response to the stressing agent. This response occurs through changes in cellular metabolism, activating its defense mechanisms that include the performance of heat shock proteins (Hsp). The objective of this work was to identify the Hsp70 family of Trichoderma asperellum and to analyze the expression of three genes encoding proteins of this family during mycoparasitism and in situations of thermal and ethanol stress. The identification of the T. asperellum Hsp70 proteins was possible from the database analysis (JGI) of the T. asperellum genome, available, but not annotated. We identified a total of 12 proteins from the Hsp70 family in T. asperellum, three of which were selected for gene expression assays. Paired cultivation was carried out between T. asperellum and phytopathogens: Sclerotinia sclerotiorum and Fusarium oxysporum in three phases of mycoparasitism: pre-contact, contact and post-contact. We verified that the expression of Tahsp70a, Tahsp70b and Tahsp70c genes varies according to the phases of the mycoparasitism and the phytopathogen studied. Expression of hsp70 under thermal stress was evaluated at the 38 °C condition for 30 minutes, 1, 2 and 4 hours and at the conditions of 4, 10 and 32 °C for 1 hour. Ethanol stress was also performed for 1 hour. During the thermal stress Tahsp70a and Tahsp70b presented higher induction and the Tahsp70c gene had its highest induction in the cold shock at 4 °C. In ethanol stress the analyzed genes did not present significant expression.
Análise do secretoma do fungo Trichoderma harzianum crescido em presença de glicose ou parede celular de Fusarium solani
(Universidade Federal de Goiás, 2010-03-31) RAMADA, Marcelo Henrique Soller; MONTEIRO, Valdirene Neves; http://lattes.cnpq.br/8264822485508916; ULHOA, Cirano Jose; http://lattes.cnpq.br/8368469162867277
Trichoderma harzianum is a saprophytic fungus, known for its potential as a biological control agent of different phytopathogens that causes losses in crops. Its action is based on different mechanisms like volatile and non-volatile antibiotics production, competition for nutrient and space, production of hydrolytic enzymes and mycoparasitism. Fusarium solani is the agent of bean dry root rot, and is responsible for significant losses. This work sought to analyze and identify secreted proteins from T. harzianum grown in the presence of F. solani cell wall, in an attempt to obtain new insights on biocontrol.In the dual culture test, T. harzianum proved to be a potent antagonist of F. solani. Some differences in the secreted proteins profile and final medium pH were observed when T. harzianum was grown in TLE medium containing glucose and in TLE and Minimum medium containing the cell wall of the phytopathogen. 33 proteins from 18 different genes were identified. The protein Sm1 was identified and secreted in all growth conditions. Many hydrolytic enzymes related to mycoparasitism, like endochitinases, β-1,3, β-1,6-glucanases, proteases and α-1,3-glicanases, were identified and among the proteases, a hypothetical metalocarboxipeptidase and a serine subtilisin-like never described for T. harzianum. Some enzymes with unkown roles in the mycoparasitism, such as β-1,3-glucanosyltransferase, secreted only in MM growth medium supernatant, were also identified.
Análise do transcriptoma de Trichoderma harzianum em resposta a exposição a cádmio
(Universidade Federal de Goiás, 2019-02-27) Oshiquiri, Letícia Harumi; Ulhoa, Cirano José; http://lattes.cnpq.br/8368469162867277; Georg, Raphaela de Castro; http://lattes.cnpq.br/3971276154067590; Georg, Raphaela de Castro; Gomes, Suely Lopes; Bailão, Alexandre Melo; Ulhoa, Cirano José
Excessive amounts of metals in soil can cause toxic effects to microorganisms, plants and animals. In contaminated regions, Trichoderma, a filamentous fungus capable of surviving in various environments, have already been isolated. The mechanisms involved in its metal tolerance are not yet fully understood, therefore, in order to evaluate Trichoderma harzianum response to cadmium, this report described the growth characteristics of the fungus and identified the gene expression profile at different concentrations of the metal, then using this data, searched for possible consensus sequences involved in its regulation. Of 13,932 genes predicted by the reference genome, 3,767 and 2,986 were differentially expressed when exposed to 1 mg.ml-1 and 2 mg.ml-1 of CdCl2, respectively. Of these genes, 2,562 were common to both treatments. Biological processes related to cellular homeostasis, transcription initiation, metabolic processes of sulfur compounds, RNA processing, protein modification and transport mediated by vesicles were up-regulated. It was observed that glutathione synthesis and its precursors’ pathways were up-regulated. Interestingly, various genes coding spliceosome components were identified. Carbohydrate, fatty acids and ionic transport metabolism were down-regulated, including important genes for the mycoparasitic potential of the fungus. Among the top down-regulated genes were genes of unknown function, therefore this analysis allowed an initial characterization of these genes. The main transcription factors related to the regulation of these genes were predicted as Cup2 (copper resistance), Cad1 (cadmium resistance) and Azf1-like (growth and proliferation). Together, the data obtained in this work suggests that the process of adaptation of T. harzianum to the presence of the metal cadmium involves the induction of several mechanisms of tolerance such as conjugation with glutathione, transport, compartmentalization in vacuoles, precipitation by sulfide, combat of ROS, DNA repair systems and quality control system for protein folding.
Análise dos níveis de poligalacturonases e glucanases expressas durante os processos de interação patogênica e saprofítica de Sclerotinia sclerotiorum
(Universidade Federal de Goiás, 2008-05-30) BARBOSA, Silvio Romero Costa; SILVA, Silvana Petrofeza da; http://lattes.cnpq.br/6823998544968373
The fungus Sclerotinia sclerotiorum can interact with a great range of vegetable species as well as to obey to the discharge especificity and patogenic specialization . It is capable to digest the cellular wall of host plants using for such a series of biochemical mechanisms and morfogenetics that optimize the invasion. Several enzymes are produced during the interaction plant-host and among them they stand out the family of the poligalacturonases (PGs) and them beta glucanases. PGs catalyze the hydrolysis of the connection glycosídic bond - 1,4 and them beta glucanases liberate glucans and oligossacarídeos during the hydrolysis. Our work tried to characterize, besides the pH, the action of these enzymes, as well as the gene expression during the interaction with bean (Phaseolus vulgaris) and under different cultivation means, pectin 1%, wall extract 1% and glucose 1%. The activities were measured by the method DNS where the amount was measured of you sugar reducers in the middle and the gene expression through electrophoretic profiles analyzed after the technique of RT-PCR. The results showed variation of the activity of PGs in the interaction being the middle with pectin with larger expression of them. Them beta 1,3 and beta 1,4 glucanases were expressed in both culture means proposed, however there was larger production of beta 1,3 during the invasion. The variation of the expression of such enzymes in different culture means suggests complexity of specific biochemical roles for your production raising new approaches for the recognition of the roads that they promote the development of the disease
Análise epidemiológica, molecular e citogenética do câncer de hipofaringe em Goiânia
(Universidade Federal de Goiás, 2006-09-29) Costa, Cesar Augusto Sam Tiago Vilanova; Cruz, Aparecido Divino da; http://lattes.cnpq.br/7868817504129985
Hypo pharynx is the under region of pharynx and serves both respiratory and digestive tract, acting as a passageway to air and food. It’s composed by squamous nonkeratinized epithelial tissue. Statistical studies indicates that 85 to 90% of neoplasies rush pyriform sinus and are characterized by fast dissemination and metastization. Epidemiological trends of hypo pharynx cancer in Goiania on period 1998 to 2003 analyzing data derived from Cancer Registry of Association for Cancer Combat of Goias, shows that the relative survival rate for men diagnosed with hypo pharynx cancer was 38% past 60 months while women presented a survival rate of 100%. Surgery was the treatments that present fewer rates (34%) and chemotherapy was associated to the most survival rate (54%). Patients that use tobacco presented a survival rate of 43% while non-tabagists present a rate of 60% of surviving. Relative survival rate for patients diagnosed with hypo pharynx tumor was 38% and estimated censure for 5 years was just 5%. Survival rate was extremely decreased due to 45% of cases were diagnosed in advanced stages of disease. It is important to point out that preventive campaign is important to evade the increasing of these pathologies that are related to behavior of tabagism and etilism. Present study also evaluates cytogenetically individuals diagnosed with hypo pharynx cancer. Cytogenetic analysis of exfoliated cells is largely used to evaluate cytotoxic and genotoxic alterations on people exposed to potentially mutagens. A number of 36 individuals were analyzed, representing case group, and 36 healthy individuals composed control group. Data was collected and analysis was performed to estimate main nuclear alterations. Standard protocols were adopted to collect and counting cells. Micronuclei test was used to determinate the frequency of cytotoxic and genotoxic alterations. Besides than micronuclei others alterations like broken-eggs, binucleated cells and piknotic nuclei are good markers of cell alterations because they are resulted from cytotoxic events frequently related to occupational exposure. Behaviors like tabagism, etilism and exposition time were also investigated to correlate cells alterations. The correlation between micronuclei frequencies of case and control group test doesn’t show statistic difference. Broken egg and binucleated cells were the most observed anomalies on case group and showed up significant statistic difference (p=0.03). Results indicates a high grade of cytotoxic anomalies on individuals with hypo pharynx cancer, specially in tabagists and chronic etilists individuals, behaviors that are correlated to induct and promote tumors in susceptible people due their capabilities to affect cell cycle. Studies about relationship between Human Papillomavirus (HPV) and cancer development are performed since 80’s. Although the correlation of HPV and cervical cancer are well established, the possible role of HPV and head and neck cancer are still unclear. Among controversial factors of HPV and carcinogenesis of squamous epithelial cells, there is a viral incidence that oscillates from 0 to almost 100% on studies that includes a myriad of viral genome detect methods, such Blotting and PCR. Importance of HPV infection and epithelial carcinogenesis is related to capabilities of High risk HPV 16 and 18 to promote cell alterations, by tumor suppressor proteins inactivation, tumor suppressor genes blockage and insertions of HPV oncogenes. On present study, HPV detection and typing was performed on tumors derived from hypo pharynx of 24 patients diagnosed with cancer (CID-O: C12,0 to C13,9). All samples was submitted to PCR with generic primers MY09/11 and GP05/06, as specific primers to HPV 6, 11, 16, 18, 33, 35, 45 and 58. Results shows that on 9 of 24 samples, HPV were detected by generic primers (a rate of 37,5%) and HPV types 16, 18 and 45 were identified, with a sample presenting coinfection of HPV 16 and 18.

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