RCAAP Repository

The Brazilian cerrado (tropical American savanna) is a semi-arid region in which plants are submitted to metabolic stress that triggers defense mechanisms when confronted with unfavorable environmental conditions. The Lauraceae is an economically important family with 52 genera and approximately 2500-2750 species consisting mostly of trees or tree-like shrubs, rich in biologically active secondary metabolites, such as lignans (sesamin, methylpiperitol and polyprenol-12), g-lactones (isoobtusilactone), alkaloids, flavonoids and terpenes, which have shown different biological and pharmacological activities. Several plants from Lauraceae family are considered endangered Brazilian cerrado species. According to studies conducted in the area of phytochemistry at the Chemical Department of the Federal University of Mato Grosso do Sul (UFMS), using the three species Aiouea trinervis, Nectandra cissiflora and Ocotea minarum several secondary metabolites have been already isolated, so the aim of the present study was to verify: i] the antioxidant activity of ethanolic extracts (EE) obtained from the leaves of Aiouea trinervis and from the leaves or stems of Nectandra cissiflora; 2] the genotoxic effects (evaluated for mutagenic and recombinagenic effects) of EE obtained from the leaves or fruits of Aiouea trinervis, from leaves or stems of N. cissiflora, and g-lactones from fruits of A. trinervis; 3] the antimutagenic effects of EE obtained from leaves of A. trinervis and from leaves or stems of N. cissiflora. The antioxidant activities were evaluated in vitro using the 2,2-diphenyl-1- picrylhydrazyl (DPPH) free radical scavenging method. The data observed with DPPH test demonstrates antiradical activity of plant extracts. The wing somatic mutation and recombination test (SMART) using Drosophila melanogaster is a short term test suited for the detection of genotoxic activity of pure compounds or complex mixtures as well as for studies on antigenotoxicity. The SMART was used to evaluate the genotoxicity of EE obtained from the leaves or fruits of A. trinervis and EE from the leaves or stem of N. cissiflora as well as of g-lactones (isoobtusilactone) isolated from fruits of A. trinervis. The extracts and the g- lactone fraction showed no mutagenic effects on spontaneous DNA lesions. Due to these preliminary observed results, EE from the leaves of A. trinervis and EE from the leaves or stem of N. cissiflora were used in combination with the free radical generator doxorubicin (DXR) (used as chemotherapeutic agent) for antigenotoxic evaluation. All in all, when EE were combined with DXR, the results generally indicated a dose-related antigenotoxic (antimutagenic) effects, which depends on to different secondary metabolites found in each type of extract, which probably operate through different mechanisms of action.

Sustainability of high yield levels on maize depends, among several factors, on the availability of high quality seeds. Resistance to processing damage is a critical factor on maize seed quality since damage during processing contributes on the quality loss during storage. The objective of this study was to identify QTLs in maize associated to seed resistance to processing damage. A 231 F2:3 segregating population derived from a cross between a resistant and a susceptible inbred to damage was screened to seed vigor after processing simulation. Levels of reduction on vigor were applied on a QTL mapping based on a 52 SSR molecular map. One QTL was identified on chromosome 9 as associated to processing damage resistance, under a LOD score of 6.56 and explaining 14% of the phenotypical variance. The genomic region identified is known as associated to physical seed traits and as containing genes related to germination process. Evidence of epistatic effects indicates that this trait involves multiple genes acting in a complex way. Similarly, the possibility of a major gene (also considering the accepted model of complete dominance) acting in recessiveness for resistance does not discard the possibility of minor genes with different effects in the degree of susceptibility. A further analyses based on differential expression may contribute on the clarification of factors involved on maize seed quality after processing.

Intracelular increase of AMP during sub maximal exercise leads to an activation of AMP deaminase following production of inosine monophosphate and ammonia. In the same direction amino acids (AAs) are used as a carbon donors for the tricarboxylic acid cycle to maintain the ATP concentration in the cell. Both metabolic pathways lead to an increase in intracellular and blood ammonia concentration. In these events, the blood ammonia concentration can raise up to 400% the resting levels. Hyperammonemia is linked with lack in neurotransmitter regulation and can be associated with neuronal excitotoxicity and/or death. Raise in ammonia synthesis during exercise is related to decrease in neuro-physical capacity in health athletes and can affect the performance. The temporary disturbances in the central nerve system caused by exercise are similar to the observed in hepatic disease and neurodegenerative disorders. Here we evaluate different exercises intensities associated with metabolic modifications induced by diet and/or supplementation to understand ammonia metabolism. We showed the blood appearance kinetics of muscle injury markers and some metabolites. We suggested that the increase in these enzymes came primarily from muscle damage instead of liver and that white blood cells are selectively mobilized independently of hemoconcentration. We also had shown the early appearance of muscle injury markers in different kinds of exercise. Our results suggest that we are able to use exercise as a general model to study ammonia metabolism in humans without requiring external ammonia exposure.

The Boophilus microplus tick is one of the most important arthropods that can parasitize bovines, causing great damages to the world livestock through direct and indirect effects. The application of chemical products is the principal method of controlling this parasite, but in function of the disadvantages of this practice, the use of vaccines is a good alternative, because they are residue free, specifics and present smaller possibility to develop resistance. With the objective of selecting synthetic peptides expressed in the capsid of phage display bacteriophages, it was produced the serum policlonal in chickens previously sensitized with total larva proteins of the Boophilus microplus tick. The peptides found in larger frequency and with larger antigenic index (bioinformatics and dot blotting with chicken serum) were selected for the immunization of mice and bovines. The initial data infer that the peptides in phagos induced immune response, evaluated by Elisa test, and the more immunogenic are: 2D7 (SNNADYKQSLL), 2E6 (VNWNSWHKTNLS), 2F3 (SIPTYTPDKVTY) and 2E4 (DAWKMRLSQMYD). The sequences selected in the present work introduce the protean motives NxxxKxxL (2D7, 2E6 and 2E4) and the motive TPDKS (2E4). These sequences presented structural similarities with some tick proteins, mainly the calreticulin. Complementary studies are being accomplished to characterize the sensibility and specificity to the selected peptides.

Ticks are implicated in serious economic losses to animal production worldwide in the order of billions of dollars. Although many antigens have been in vaccine tests, none has been effective in the control of ticks, which justifies the development of new antigens as well as new vaccine strategies. Phage Display techniques have been widely employed to map epitope structures, which have served as the basis for developing molecular vaccines. In the present study, we have applied this technique to map specific epitopes of Rhipichephalus (Boophilus) microplus and to validate the peptides as potential immunogens, we have adopted a process of selection prior to final field tests. This strategy was established in order to reduce the number of clones to be tested in the field. Six Phage -displayed random peptide libraries were selected with seven different strategies against the purified hyperimmune serum of chickens (IgY) that were immunized with total proteins of larvae and adults of Rhipicephalus (Boophilus) microplus. The selected Phage clones were sequenced, translated and analyzed through bioinformatics. To evaluate the potential of these phagotopes as effective candidate vaccines, ELISA assays, dot-blot, mice immunization (MI), humoral immune response (HIR) of cattle against the clones and a cutaneous hypersensitivity tests in cattle were performed. The selected Phage clones were analyzed through bioinformatics, generating 107 different peptides in a total of 281 sequences. Some selected phagotopes showed excellent matches with linear sequences of known proteins of the Rhipicephalus (Boophilus) microplus. Peptides that did not present significant matches with known proteins, but shared extensive homology among each other, were clustered and classified as conformational epitopes of Rhipicephalus (Boophilus) microplus, or considered as mimotopes of unknown proteic antigens. Among all clones tested by dot-blots, the 40 most reactive ones were further screened by HIR and MI. The mice sera raised against the Phage clones clearly recognized tick proteins indicating that the phagotope-induced immune responses were antigen-specific, but not all could be identified by the sera of naturally infested cattle (Bos taurus and Bos indicus). Four clones were then selected to go through the cutaneous hypersensitivity tests, and apparently all of them were effectives at diferent degrees. The stringent selection processes coupled with this schemes of validation assays allowed us to narrow down the number of peptides that should be tested in the field. One of the advantages of using whole recombinant M13 virus, carrying the recombinant peptide in fusion with the pIII protein of the virus, is that its capsid acts as adjuvant and could be tested directly without any further mixture. Additionally, we have finally used anergy skin tests (cutaneous hypersensitivity tests) once they are sometimes used to guide clinical decisions. We have assumed that cutaneous hypersensitivity tests measure a common property of cellular immune function relevant to the outcome, which together with the humoral response in naturally infested animals may provide us with sequences of peptides that may be relevant as vaccines. Finally, we have used those sequences to produce multiple antigen peptide system (MAPS), which will be used in future field tests. The present work demonstrates that the whole epitope profile can obtained through screening the Phage Displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitopedisplaying Phage s as peptide vaccines against ticks.

The continuous investigation on gene polymorphisms may lead to the discovery of important associations with quantitative traits, explaining part of the existing variability. Considering the essential role of the somatotropic axis in the regulation of lactation, the purpose of this study was to associate polymorphisms of six genes: growth hormone releasing hormone (GHRH), growth hormone releasing hormone receptor (GHRHR), pituitary specific transcription factor 1 (Pit1), growth hormone (GH), insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), with production traits of dairy cattle, such as: total milk production, average daily milk production and lactation period, analyzing the effects of gene interactions in bovine &#8541; Girolando. Genotyping of the 356 females was accomplished by the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) technique. Statistical analyses were performed by using a mixed model and the averages grouped by genotype were compared by the Student t test. Results revealed a superiority of the heterozygote (GHRH/HaeIII) to total production (p < 0.01) and average daily milk production (p < 0.05). Favorable effects (p < 0.05) of the BB genotype compared to AA (Pit1/HinfI) and CC in relation to DD (GH/MspI) were also verified in the milk production. Additive effects of allele B (Pit1/HinfI) and C (GH/MspI) were observed in the milk production, generating an increase in the lactation of 116.2 kg and 68.51 kg of milk, respectively, for each incorporated allele. Through the analysis of gene interactions, a superiority (p < 0.05) of genotype BB (Pit1) in the presence of AA (IGF1) was demonstrated in total and average daily milk production. A shorter lactation period (p < 0.05) was still found in individuals with the genotypic combinations AA(Pit1)/BB(GHRHR), BB(Pit1)/AA(GHRHR), AB(IGF1)/AA(GHRH). The importance of gene polymorphisms in the GH pathway identified in this study suggests that these genes may become potential molecular markers to assist the selection of the Girolando breed as an auxiliary tool in genetic improvement programs.

This research aimed at the estimation of genetic and statistical parameters as well as the determination of genetic and genotypic values utilized for selection among and within hybrid populations of Cacao (Theobroma cacao L.). The data utilized were taken of individual plants in two field experiments of competition of hybrid combinations installed in proper experimental design and conducted at the experimental farm Paulo Dias Morelli (CEPLAC) located at the main producing region of the Brazilian Amazon basin. Due to the unbalance of the original set of data, a peculiarity of the perennial habit of the species, it was used the REML/BLUP, an mixed model. The variable in study was the total number of harvested fruits per plant taken for three consecutive years from one year before through one year immediately after the climax. The parameters repeatability and heritability were estimated based on the estimates of phenotypic and genotypic variances. Similarly the breeding values of the hybrids and of the individuals were estimated. The repeatability values found allowed to infer, with 95% precision, on the number of measurements needed to discriminate among hybrids at plot level (seven for experiment 1 and six for experiment 2). Such numbers are smaller than the ones really utilized in both experiments. Furthermore, four to five seasons were enough to discriminate amongst hybrids. With the level of precision of 80%, both the number of measurements and seasons were three, which is practically acceptable. The analyses for heritability, genetic and genotypic values (BLUP) were performed for both the individual experiments and the combined trials. Heritabilities estimates were of low magnitude (0.20 to 0.21 for hybrids and 0.13 to 0.19 on individual plant basis. High significant estimates were found, however, for all the estimates of genetic and genotypic variances, regardless of the type of analysis. The results obtained for both analyses individual and combined, on discrimination by hybrids and individual plant BLUP were similar as to the hybrids selected and their rank, as well as to the number of selected families regarding both breeding simulations on the short and on the medium range. With regard to the discrimination among individual plants there was a slight discrepancy among the different analyses. According with the combined analysis, three genotypes would be excluded (one in experiment 2) and fourteen extra genotypes would be selected in experiment 1 (two of those of the same hybrid that had their genotypes excluded). It was concluded that the combined analysis was adequate as well as the REML/BLUP procedure for the selections of hybrids per se and genotypes. These results as far as selection of hybrids per are is concerned, indicate the strong necessity of performing evaluations in all the field experiments that have in their composition the hybrid combinations recommended for commercial plantations applying the REML/BLUP methods with data taken on an individual plant basis and with number of measurements adequately repeated (plots and seasons). In the case of non-selected hybrids on the present study, that in turn are presently recommended, they should be eliminated of the hybrid mixtures offered to the growers. They should be also substituted by seeds of the two synthetic varieties which may be immediately produced from the selected individual plants in their respective experiments.

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CHAPTER II: Physical exercise induces biochemical changes in the body that modify analytes in blood and saliva among other body fluids. Many of these salivary protein analytes and their functions remain uncharacterized. This study analyzed the effect of an incremental effort test on the salivary protein profile to determine whether any specific protein is altered in response to such stress. We also measured thresholds of salivary alpha amylase, total salivary protein and blood lactate and searched for correlations among them. Twelve male cyclists underwent a progressive test in which blood and saliva samples were collected simultaneously at each stage. The salivary total protein profile revealed that physical exercise primarily affects the polypeptide corresponding to salivary alpha-amylase, the concentration of which increased markedly during the test. We observed thresholds of salivary alpha-amylase (sAAT), total salivary protein (PAT) and blood lactate (BLT) in 58%, 83% and 100% of our sample, respectively. Pearson s correlation indicates,during the test, a strong and significant association between sAAT and BLT (r= 0.84 p<0.05), sAAT and PAT (r= 0.83 p<0.05) and BLT and PAT (r= 0.90 p<0.05) . The increased expression of the salivary alpha-amylase (sAA) polypeptide suggests that sAA is the main protein responsible for the increase in total protein concentration of whole saliva. Therefore, monitoring total protein concentration is an efficient tool and an alternative noninvasive biochemical method for determining exercise intensity.

CHAPTER I: Early detection of oral squamous cell carcinoma (OSCC) is an important factor to reduce mortality rates and treatment success is directly related to its diagnosis at early stages. However, unfortunately, most oral carcinomas are diagnosed in late stages of the disease. This is the first study that describes an scFv fragment, selected by Phage Display against total salivary proteins, that effectively recognize antigens with diagnostic potential for early detection of oral cancer. ELISA immunoassays in 30 samples of saliva from OSCC patients and 30 healthy subjects showed that the scFv D09 recognize antigens present in tumoral samples (p <0.0001). The diagnostic test showed high discriminative power with a significant ROC curve (AUC=0.97) and 100% specificity. Imunohistochemistry tests revealed that the selected antibody also showed higher affinity to tissues related to well differentiated oral carcinomas, such as keratin pearls. The two-dimensional proteomic analysis revealed that the scFv D09 recognizes two spots in saliva samples (~50 kDa; pI 5.75 e 5.86) and only one in tissue samples (~28 kDa and pI 4.68). Final characterization of the peptides sequences is under investigation through mass spectrometry. These data, although preliminary, may significantly alter the diagnosis of oral cancer, once they provide strong evidence that saliva is a powerful clinical tool for early detection through a non-invasive and highly sensitive diagnostic method. CHAPTER II: Several studies have been suggesting annexin A1 protein as an active player in tumorigenesis of many organs. Nevertheless, its tumor biomarker role has been mainly studied in tissues by immunohistochemistry or cell culture. Hence, in this investigation, the peripheral blood from 27 oral squamous cell carcinoma (OSCC) patients and 25 negative control individuals were examined by quantitative real-time PCR. Down-regulated ANXA1 expression at mRNA level was observed in OSCC samples (p = 0.026). Significantly diminished mRNA levels correlated to age, sex and the anatomical site of the tumor lesion were observed. Moreover, the ROC curve analysis revealed the performance of ANXA1 expression as a suitable biomarker for patients with oral cavity cancer, especially those with 60 years of age or older and/or women. For the first time, ANXA1 mRNA is revealed as blood-based biomarker, and its adoption for complementary non-invasive diagnosis of OSCC is suggested. These results suggest that, beyond the anti-inflammatory function, annexin A1 may also play a tumor suppressor role in peripheral blood cells, such as leukocytes.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Centro Universitário de Patos de Minas

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

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CHAPTER II: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography using DEAE-Sephacel and gel filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS PAGE under reducing conditions and gave one main peak at 29814 kDa in MALDITOF/ TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the A&#945;-chain of fibrinogen and, more slowly, the Bß-chain. Its fibrinogenolytic activity is inhibited by b-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at 80 °C. Intraplantar injection of Eumiliin (1 25 &#956;g/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1 - 5 h after enzyme injection. Intraplantar injection of Eumiliin (1 25 &#956;g/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. CHAPTER III: A protease, which we designate Eumiliin, was isolated from the latex of Euphorbia milii var. hislopii by a combination of ion-exchange chromatography using DEAE-Sephacel and gel filtration with Sephadex G-75. Eumiliin is a monomeric protein with an apparent molecular mass of 30 kDa by SDS PAGE under reducing conditions and gave one main peak at 29814 kDa in MALDITOF/ TOF mass spectrometry. Eumiliin has caseinolytic and fibrinogenolytic activities, but no hemorrhagic or defibrinating activities. The enzyme readily hydrolyzes the A-chain of fibrinogen and, more slowly, the B-chain. Its fibrinogenolytic activity is inhibited by b-mercaptoethanol and leupeptin. In contrast, EDTA and benzamidine did not affect the activity of Eumiliin. The caseinolytic activity of Eumiliin had a pH optimum of 8.0 and was stable in solution at up to 40 °C; activity was completely lost at 80 °C. Intraplantar injection of Eumiliin (1 25 &#956;g/paw) caused a dose- and time-dependent hyperalgesia, which peaked 1 - 5 h after enzyme injection. Intraplantar injection of Eumiliin (1 25 &#956;g/paw) also caused an oedematogenic response that was maximal after 1 h. Morphological analyses indicated that Eumiliin induced an intense myonecrosis, with visible leukocyte infiltrate and damaged muscle cells 24 h after injection. CHAPTER IV: Bhalternin and Eumiliin are proteases extracted from the venom of Bothrops alternatus and latex of Euphorbia milli var. hislopii, respectively. The stabilities of Bhalternin and Eumiliin against denaturation by heat and urea were determined and compared. The plant protease proved to be tougher when heated at high temperatures and also when subjected to the action of urea as denaturant. Further studies are needed to better characterize the conditions for stabilization of these enzymes, especially those related to the strategies needed to protect the transport and storage in the commercial, therapeutic and biotechnological processes.

Universidade Federal de Uberlândia

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

Concentration of ammonia in blood increases during endurance exercise and can be toxic for muscle cells and brain metabolism, potentially leading to both peripheral and central fatigue. Intense exercise can promote failure in replace ATP leading to greater deamination of AMP and consequent increase in blood ammonia. High concentration of ammonia is toxic and harms the performance. Keto acids has been proposed to capture the blood nitrogen compounds. Here we describe the protective effects of acute and chronic keto acids supplementation on nitrogen metabolism and physical performance in male rats submitted to a resistance training protocol. This study investigated the protective effect of supplementation keto analogues against acute nitrogen compounds and physical performance in rats submitted to exercise.

Ammonia (here used as a synonym for NH3 + NH4+) is a toxic metabolite with deleterious effects on the central nervous system. Exercise can be used as a model to study ammonia metabolism and hyperammonemia. The temporary disturbances in the central nerve system caused by exercise are similar to the observed in hepatic disease and neurodegenerative disorders. Increase of adenosine monophosphate (AMP) during prolonged exercise leads to an production of inosine monophosphate (IMP). Furthermore, amino acids are used as carbon donors for the tricarboxylic acid cycle to maintain the ATP concentration in the cell. Both metabolic pathways lead to an increase in intracellular and ammonemia concentration. In these events, the blood ammonia concentration can raise up to 400% the resting levels. Changes in ammonia levels in response to exercise can be managed through the use of amino acids or carbohydrates that interfere with the metabolism of ammonia. Low carbohydrates diet (called here as ketogenic diet) combined with physical exercise can reduce glycogen stores, inducing early states of hyperammonemia. We explored the ability of a ketogenic diet to enhance the effect of exercise on ammonia production. Though, keto analogues can serve as a nutritional supplement to provide amino acids of high biological value, as well as a tool for ammonia sequestering. In the present study, we used exercise stress to investigate ammonia metabolism. We studied a low-carbohydrate ketogenic diet and exercise as a hyperammonemia model in order to understand the role of the association of keto analogues and amino acid (KAAA) supplementation in ammonia metabolism.