RCAAP Repository

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Universidade de Franca

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Diabetes mellitus is a chronic disease that causes hyperglycemia due to deficiency in insulin production or ineffective use of the insulin that is produced. Phaseolamin is a glycoprotein extracted from Phaseolus vulgaris seeds accountable for inhibiting the activity of the enzyme alpha-amylase. Phaseolamin has been marketed and prescribed for reducing blood glucose and body weight in humans, although there are contradictions as to their effectiveness. On the other hand, Vochysia drums popularly known as \"sweet corner\" has been used in folk medicine to treat diabetes mellitus type 1 and type 2 in Uberlandia, Brazil. The mechanism of action and the phytochemical profile of the studied species is unknown. The aim of this study was to evaluate the anti-diabetic and antioxidant potential of V. rufa extract (V) and a sample of commercial phaseolamine (P) on the biochemical, antioxidants and histological parameters in non-diabetic rats (DN) and diabetic streptozotocin-induced (D) mice for 43 days. The primary drums V. phytochemical extract showed the presence of proteins and triterpenoids. The animals received water (ND control groups and D), V drums extract (500 mg / kg) (NDV and DV groups), phaseolamine (500 mg / kg) (NDP and PD group) and glyburide (6 mg / kg) (NDG and DG groups) and acarbose (25mg / kg) (NDA and DA groups) as a control drug, respectively, will gavage. DV groups, NDP, DV and DP showed no reduction in body weight compared to control groups (glibenclamide, water and acarbose). However, NDV, NDP, DV and DP, as well as NDG DG rats and reduced glucose levels compared before and after treatment. Group NDV increased urea levels (p <0.05) and aspartate aminotransferase (AST) (P <0.001) compared to control. DV compared to group D decreased the levels of AST (P <0.05), ALT (P <0.05) and HDL-C (P <0.05) as compared to group D, DG increased acid levels uric (P <0.01), creatinine (P <0.05) and decreased alkaline phosphatase levels (ALP) (P <0.01). SD rats significantly increased uric acid levels (P <0.01), creatinine (P <0.05) and urea (P <0.01) compared with mice the levels of D. The level of alkaline phosphatase (ALP) (P <0.05) decreased significantly in PD rats compared with rats D. NDP similar to the NDA increased levels of urea (P <0.01) and ALT (p <0.05). Therefore, the extracto of Vochysia was able to reduce blood glucose and alleviated the renal and hepatic effects observed in 5 streptozotocin-induced diabetic rats as well as the sample phaseolamine can cause damage to liver and kidney functions.

CHAPTER I - Coccidioidomycosis, known as Valley Fever , is a disease caused by the fungus C. immitis/ C. posadasii. This disease is endemic to the Southwestern United States. Due to the complexity of the disease, multiple tests, such as serological, microbiological and microscopical, should be performed to identify this mycosis. Because of that we have proposed a new approach for the development of novel biomarkers, which is based on the subtractive proteomic strategy known as Phage Display to identify ligands that mimics antigenic mimotopes. Interestingly, the same strategy was used to select antibody fragments, such as scFv (single chain fragment variable) molecules, which may be used to block or neutralize a microorganism, reinforcing its potential use as therapeutic molecules. We showed that one of our selected scFv, clone B8, had high specificity and sensitivity with significant difference between chronic and healthy non-immune patients. Several clones from the peptide biopanning were able to significantly distinguish between purified IgG from chronic patients and healthy non-immune individuals. This investigation brought important perspectives for the diagnosis and therapeutics of the disease. CHAPTER II - Coccidioidomycosis is caused by the pathogenic dimorphic soil-dwelling fungus of the genus Coccidioides. The lack of a thorough understanding of its immune response guided us to investigate the antigen presenting cells profile, and we have successfully demonstrated that specific cell lineages (CD3-CD19- CD14+/-CD11c+/-) present differential expression of receptors and cytokine production. Under stimulation of endospores and spherules, healthy-immune patients presented higher levels of IL-10, and were similar to the chronic patients. We have also observed a significant reduction of PRRs surface expression in CD14+CD11c- cells, such as TLR2, CD206 (Mannose Receptor) and Dectin-1. TLR2 expression from HLA-DR+CD14-CD11c+, HLA-DR+CD14+ and CD14+CD11c+ cells of chronic patients were significantly decreased under same stimuli, which maybe is related with internalization of the receptor after phagocytosis of the fungus.

The innate immune system plays as the first line of defense of all animals. It possesses cells and molecules that act in fighting many types of pathogens. The defense cells carry recognition receptors, which, trigger reactions for the production of molecules that will combat the pathogens. The NF-kB pathway is a pathway conserved in animals, it is part of this system and possesses two important molecules for its operation, MyD88 and Tollip. The role of MyD88 protein is coupling together with other proteins and transmit the signal inside the cell, while Tollip functions as a negative regulator at the end of the same pathway. These two protein sequences were analyzed in different animal species, their evolutionary aspects were studied among the domains of the MyD88 protein, and between the complete sequences and conserved parts of the Tollip. Using bioinformatics tools, we can see that Tollip has successive modifications to optimize its stabilization accumulating aliphatic residues. Differences were found between the stability of primates and arthropods proteins. In addition to the Tollip showing the need for ion Ca2 + for the maintenance of the cavity, it was possible to identify a ligand 768 probably related to inhibition of Tollip. In MyD88 protein we identified a greater identity in sequences of primates in relation to other species analyzed, also it was enhanced the high similarity of some amino acids interface with the functionality of the protein. The TIR domain has demonstrated greater identity between the fields of protein and a greater MydD88 ancestry of its sequence regarding the MD region, which may suggest a relationship with the largest number of interaction partners.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais

In this work we describe the isolation of a new L-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity and hydrophobic chromatography steps. BpirLAAO-I is a homodimeric acidic glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively), that displays a high specificity towards hydrophobic/aromatic amino acid residues while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a similarity and other LAAOs from: Bothrops spp, Crotalus spp, Calloselasma rhosostoma, Agkistrodon spp, Trimeresurus spp, Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.

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Chapter II - [Introduction] The stability of a biological membrane is resultant from its composition and from the composition of the internal medium of the living organism, which depends on genetic influences, feeding and life style. [Objective] This work aimed to study transversally in a human female population the dependence of the erythrocytes stability with age, nutritional status, erythrocytes counting and medium corpuscular volume (MCV). [Subjects] The sample was constituted of human females (N = 67), with 20 to 94 years, proceeding from a defined region with similar dietary habits and life styles. [Methods] The erythrocytes stability against hypotonic lysis and ethanol action was given by the concentration of NaCl (H50) or ethanol (D50) capable to promote 50% of hemolysis. The H50 values were determined at 26, 32, 37, 42 and 47 °C; the D50 values were determined only at 37 °C. The nutritional status of the volunteers was evaluated with the Mini-Nutritional Assessment (MNA). [Results] H50 was inversely dependent (P<0.05) with age and MCV, but not with MNA score neither the erythrocyte counting of the volunteer, at 26, 32, 37 and 42 °C, but not at 47 °C. D50 presented a linear and positive dependency with the volunteer age. H50 present a linear and negative dependence with the temperature increase. The H50 values in the thermal dependence line of H50 for the females with 20 to 39 years were significantly higher than those for the females above 60 years. [Conclusions] The stability of the erythrocytes against hypotonic stress and denaturation by ethanol increased with the age of the volunteers. This stability increase was not determined by differences in the nutritional status neither in medium corpuscular volume. The stability of the erythrocytes decreased significantly with the temperature increase between 26 and 47 °C. The females above 60 years presented erythrocytes with higher stability than the females with 20 to 39 years within this thermal interval.

Conselho Nacional de Desenvolvimento Científico e Tecnológico

CAPÍTULO I: The amplification of fragmentos scFv (single chain fragments variable) of immunized animals is an important method to generation of monoclonal antibodies. In this work chickens had been immunized with proteins of the larval and adult phases of the Boophilus microplus for the generation of an antibodies combinatorial library. Total RNA of spleen had been extracted and amplification reactions (PCR) had been made using specific primers of the antibodies variable regions with the purpose to get all the variability of the immune repertoire in the construction of the library. Five cycles of selection (biopanning) had been made for capture of clones scFv reactive to total proteins of larvae and adults ticks. The affinity of clones to proteins was confirmed by ELISA demonstrating an increase of the affinity of clones to the ligante throughout the five cycles of selection. The sequencing of clones demonstrated a predominance of identical clones for the library using both larvae and adults proteins. The recombinant antibodies had been used for selection and characterization of epitope to proteins of the catle tick for use as vaccine in the control of this parasite. CAPÍTULO II: With the objective of selecting and characterizing new ticks vaccine targets, we developed a combinatorial antibody fragment library (scFv), expressed in the capsid of bacteriophages, produced from tick larval and adult protein immunized chicken Ig repertoire These antibodies (scFv) recognize a protein of the total larval and adult s extract of the proximally 80 Kd, by western blotting tests, and the sequence of these reactive proteins was checked by N-terminal sequencing. The band corresponded to a GP80 protein with high similarity and is found in huge quantities in parasite eggs and larval stages. With the objective of selecting mimotopes of B. microplus by phage displayed epitope characterization, the recombinant antibodies with major frequency were submitted to a selection against a constricted peptide library displayed on phages. The cross reactivity was confirmed by the recognition of one of those recombinant antibodes (scFv) to a motif similar to GP80 sequence and others peptide (Serotonin receptor, Notch-like protein, Paramiosin and Antigen B membrane) obtained from peptide library selection. This work confirms the GP80 as a vaccine candidate and points out the usefulness of phage display methodology for selection of new represents the selection efficiency of new B. microplus.vaccine targets.

CHAPER I: Through a review of the literature, this article discusses the genetic mechanisms that control tooth morphogenesis. Emphasis is placed upon the structure and function of some key molecules that participate in interactions between its epithelial-mesenchimal components. In this paper we will can understand the mechanisms that control tooth morphogenesis and the dentistry should pay special attention to possible consequences of tooth number anomalies. CHAPER II: The analysis of DNA is widely employed in the genetic studies. Human DNA in most cases is performed with samples obtained from peripheral blood. The use of buccal epithelial cells as a source of DNA for PCR amplifications has several advantages over blood sampling. In the present study our objective was to standardize DNA extraction from an oral swab, using a simple method. To test DNA quality, we amplified the exons 2 of MSX1 gene and the promoter region of LEF1 gene to patients with hypodontia. In conclusion, we standardized a simple DNA. extraction of oral cells, which presented lower costs and faster results, indicating to that DNA from oral brushes/swabs are a reliable source for genetic studies. The quantity and quality of extracted DNA was shown to be adequate for PCR and polymorphism analyses. CHAPER III: Hypodontia, the congenital absence of one or a few teeth, is one of the most common alterations of the human dentition. The most common permanent missing teeth are the third molars, second premolars, and maxillary lateral incisors. Although hypodontia does not represent a serious public health problem, it may cause masticatory and speech dysfunctions and esthetic problems. In human the participation of MSX1 gene in craniofacial development have been evidenced by the studes that showed mutations in this gene. Hypodontia were shown to be caused by mutations in the MSX1 gene in human however, the mutation in the MSX1 gene cannot explain all types of tooth agenesis. Our data suggest that polymorphisms in MSX1 gene are associated with hypodontia.

The major objectives of this study were to evaluate phenotypical adaptability and stability of soybean genotypes in the state of Mato Grosso, using different methodologies and to identify soybean genotypes with partial resistance to Asian rust (Phakopshora pachyrhizi) under artificial inoculation. Phenotypical adaptability and stability were evaluated in three subsequent years (2003/04, 2004/05, 2005/06), two trials, divided into culture cycle (semi-early/ medium and semi-late/late), in counties of the state of Mato Grosso, using as experimental design randomized blocks, with three repetitions. All genotypes belonged to the breeding program of UFU. Joint analyses were done with genotype x planting location and genotype x planting location x harvest, and the averages were compared by the Scott Knott and Tukey tests for subsequent computation of phenotypical adaptability and stability by seven different methods: multivariate method AMMI, reability index; bi-segmented linear regression method; simple linear regression; deviation of the ideal maximum method, variance components method, and method of ecovalence. The experiment to evaluate partial resistance to Phakopsora pachyrhizi was done in Uberlândia-MG, in a green-house, from December 2004 to February 2005 and consisted of three evaluation periods. he following resistance characteristics was evaluated: average latent period (PLM), average number of pustules per leaflet and rust severity. Based on the variables average number of pustules per leaflet and rust severity the area under the disease progress curve was calculated. Subsequently, analysis of variance was done and the averages compared by the Scott Knott test, at 5% probability. The methodologies studied for phenotypical adaptability and stability were similar and complemented each other for the results obtained. The environments differed in favorability as a function of agricultural year. In the early trials, harvest 2003/04, the most productive materials were genotypes UFU 01, Msoy 8400 and Emgopa 316; in 2004/05 was line UFU 19, while in 2005/06 was UFU 13, 18, 22, 23, 24, 27, 28, 29, 35, 36 and Msoy 8585. The lines UFU 23 (harvest 2004/05) and UFU 24 (harvest 2005/06) presented phenotypicial adaptability and stability in all methodologies evaluated. As for the semi-late/late cycle trial a greater performance was found for line UFU 21 (Confiança x Xingu) in the harvest 2004/05 and the materials UFU 18 (FT 50.268-M x Msoy 8400) and 28 (IAC 8-2 x IAC 100), in the harvest 2005/06. Partial resistance to Asian rust was found in genotypes P 5001 and Coodetec 78. The variables studied can be recommended for epidemiology studies of the pathosystem soybean x P. pachyrhizi. Cluster analysis allowed grouping genotypes with partial resistance to Asian rust.

CHAPTER II: The infection for T. gondii affects more than a third of the world population, affecting mainly imunocompromised individuals. Therapies in use are limited because of parasite resistance and side effects. Then, the search for new drugs to control infection is extremly important. The effects of neuwiedase, an isolated metaloprotease from Bothrops pauloensis venom, on the in vitro invasion and proliferation of the T. gondii in human fibroblastos.was investigated The treatment was done on cells previously infected and on the parasite before the infection, and results showed infection inhibition levels of 71% and 61%, respectively. The mechanisms involved in neuwiedase action need to be better investigated, but the capacity of enzyme to degraded some extracelular matrix components such as laminin, fibronectin and collagen type I can be important in the reduction of host cells invasion by T. gondii . CHAPTER III: The release of pro-inflammatory cytokines and chemokine (IL-12, TNF-α and IL-8) and anti-inflammatory cytokine (IL-10) from human peripheral blood mononuclear cells (PBMC) was evaluated after exposure to neuwiedase, a Zn2+ dependent metaloprotease isolated from Bothrops pauloensis venom. Initially, using the MTT metabolization assay, we show that in all concentrations tested (3, 6 e 12 μg/mL), neuwiedase did not affect PBMCs viability. Suppernatants of PBMC culture in presence of neuwiedase were collected and cytokine and chemokine levels were determined by sandwich ELISA. Under neuwiedase stimulation, PBMCs significantly increased the production of IL-12p40, IL-8 and TNF-α but not IL-10 production compared cells to not stimulated with neuwiedase. This study confirms the inflammatory properties of neuwiedase involving the liberation of proinflammatory mediators, probably contributing to the severe local damage induced by the Bothrops pauloensis venon. CHAPTER III: The release of pro-inflammatory cytokines and chemokine (IL-12, TNF-α and IL-8) and anti-inflammatory cytokine (IL-10) from human peripheral blood mononuclear cells (PBMC) was evaluated after exposure to neuwiedase, a Zn2+ dependent metaloprotease isolated from Bothrops pauloensis venom. Initially, using the MTT metabolization assay, we show that in all concentrations tested (3, 6 e 12 μg/mL), neuwiedase did not affect PBMCs viability. Suppernatants of PBMC culture in presence of neuwiedase were collected and cytokine and chemokine levels were determined by sandwich ELISA. Under neuwiedase stimulation, PBMCs significantly increased the production of IL-12p40, IL-8 and TNF-α but not IL-10 production compared cells to not stimulated with neuwiedase. This study confirms the inflammatory properties of neuwiedase involving the liberation of proinflammatory mediators, probably contributing to the severe local damage induced by the Bothrops pauloensis venon.

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CHAPTER I: Pachymerus nucleorum Fabricius 1792 is a beetle specie that predate babaçu coconut (Orbignya sp.). It belong to Bruchinae coleoptera subfamily, whose representants are know as seed bits. We found a precipitated fraction from larvae of P. nucleorum that is not solubilized by Triton X-100 (P3) and presents Ca2+-ATPase and K+/EDTA-ATPase activity. The objective of this work was solubilize and characterize the enzyme responsible for the Ca2+-ATPase activity. The Ca2+-ATPase was partially solubilized with 600 mM NaCl and the soluble fraction from this assay (S4) was better characterized. We noticed in SDS-PAGE a strongly stained polypeptide with 205 kDa and two others with 130 and 102 kDa. 600 mM NaCl inhibits about 40% of P3 Ca2+-ATPase activity, and 84% with 2 M NaCl. We found in S4 Mn2+-ATPasic activity, corresponding to 40% of the observed with calcium, and no Mg2+-ATPase activity was detected. In presence of Zn2+ or Fe2+ the ATPase activity was null and when incubated with cobalt, copper, barium, litium or Fe 2+, the ATPase activity correspond to less than 20% of the Ca2+-ATPase activity. The Ca2+- ATPase activity was completely inhibited by magnesium, copper ou zinc. Cobalt, manganese, Fe3+ or Fe2+ inhibited in more than 60% such activity, and barium and litium didn t affect the calcium-dependent activity. In this work, the Ca2+-ATPase activity of Pachymerus nucleorum larvae was partially solubilized with 600 mM NaCl and presents Ca2+-ATPase activity inhibited by magnesium. CHAPTER II: Ca2+-ATPases are ATPases involved in transport of calcium across membranes. Sarcoplasmic and endoplasmic Ca2+-ATPases are irreversibly inhibited by thapsigargin in micromolar concentrations. The objective of this work was to test the effect of thapsigargin on Ca2+-ATPase activity isolated from Pachymerus nucleorum larvae, a beetle especie that belongs to Bruchinae coleoptera subfamily. The P. nucleorum larvae were homogeneized and the precipitated fraction (P3 fraction) not solubilized with Triton X-100 was used to thapsigargin inhibtion tests. Thapsigargin 140 &#956;M inhibit about 80% and 90% of Ca2+-ATPase activity of P3, when added in the begin or 5 minutes before the start reaction, respectivelly. Preincubation with ATP or calcium partially prevented the inhibition of Ca2+-ATPase activity. The inhibition was completely prevented with pre-incubation with both ATP e calcium. The results showed in this work suggest that the ATPase isolated from P. Nucleorum larvae treats of a Ca2+-ATPase.