Detecção de Chlamydia trachomatis em amostras cervicais por reação em cadeia da polimerase

Chlamydia trachomatis é o agente causal de uma das infecções sexualmente transmissíveis (IST) mais prevalentes da atualidade. Os maiores problemas no controle desta IST estão no caráter assintomático da infecção e no seu difícil diagnóstico laboratorial. Com o advento dos testes moleculares, grandes avanços ocorreram na área do diagnóstico laboratorial da infecção clamidial. O presente trabalho teve por objetivo desenvolver um método de detecção de C. trachomatis por PCR a partir de amostras cérvico-vaginais. A seqüência alvo escolhida para amplificação consiste de um segmento da ORF 4 do plasmídio críptico de ocorrência natural nesta bactéria. Noventa e duas amostras cérvico-vaginais foram submetidas ao protocolo de PCR in house proposto. Os produtos de PCR foram detectados por visualização direta após eletroforese em gel de agarose com brometo de etídio e por exposição radiográfica após hibridização com sonda homóloga. As amostras foram testadas paralelamente pelo método de captura híbrida para detecção de C. trachomatis. O kit COBAS Amplicor (Roche) foi utilizado para resolver resultados discrepantes. A seqüência do fragmento de 201pb foi confirmada por clivagem enzimática e por seqüenciamento. O teste de especificidade dos primers confirmou especificidade dos mesmos frente ao DNA de diferentes agentes patogênicos e da flora normal feminina. Do total de amostras analisadas, 50 foram positivas por captura híbrida, 51 foram positivas por PCR in house e 67 positivaram após hibridização.O teste de McNemar indicou haver concordância entre os métodos analisados dois a dois (P<0,001). Verificou-se concordância moderada nos comparativos entre captura híbrida e PCR (valor de Kappa: 0,45; DP 0,093), captura híbrida e hibridização (valor de kappa: 0,389; DP 0,091) e, PCR e hibridização (valor de Kappa: 0,634: DP 0,077). O método de PCR in house proposto para a detecção de C. trachomatis é uma técnica rápida e de baixo custo para o diagnóstico, controle e monitoramento dos casos da infecção. Estudos complementares, no entanto, são necessários para implementação deste teste em laboratórios da rede pública.

Introdução a disciplina de Engenharia de Software

Esta videoaula é uma apresentação do conteúdo que será trabalhado durante a disciplina de Engenharia de Software. O objetivo principal da disciplina é ensinar os alunos a projetar um sistema orientado a objetos usando a linguagem de modelagem UML. O objetivo secundário é ensiná-los a desenvolver um sistema de forma iterativa e incremental. Os diagramas abordados durante o curso são Casos de Uso, Diagramas Conceituais e de Classes, Diagrama de Seqüência, Diagrama de Colaboração e de Estado. Para exemplificar e transmitir tais ensinamentos será usado um sistema de reserva de uma rede de hotéis. Este material é voltado aos estudantes de graduação que estão cursando disciplinas de Engenharia de Software.

Determinantes genéticos da hanseníase em uma população do Rio Grande do Norte

Background: Leprosy can cause severe disability and disfigurement and is still a major health in different parts of the world. Only a subset of those individuals exposed to the pathogen will go on to develop clinical disease and there is a broad clinical spectrum amongst leprosy patients. The outcome of infection is in part due to host genes that influence control of the initial infection and the host´s immune response to that infection. Aim: Evaluate if polymorphisms type SNP in the 17q118q21 chromosomic region contribute to development of leprosy in Rio Grande do Norte population. Material and methods: A sample composed of 215 leprosy patients and 229 controls drawn from the same population were genotyped by using a Snapshot assay for eight genes (NOS2A, CCL18, CRLF3, CCL23, TNFAIP1, STAT5B, CCR7 and CSF3) located in chromosomic region 17q118q21. The genotype and allele frequency were measured and statistical analysis was performed by chi-square in SPSS version 15 and graph prism pad version 4 software. Results: Ours results indicated that the markers NOS2A8277, NOS2A8rs16949, CCR78rs11574663 and CSF38rs2227322 presented strong association with leprosy and their risk genotype were GG, TT, AA and GG respectively. The risk genotypes for all markers associated to leprosy presented recessive inheritance standard. When we compared the interaction among the markers in different combination we find that the marker NOS2A8277 associated with CCR78rs11574663 presented highest risk probability to development of leprosy. When we evaluated the haplotype of the risk markers it was found a haplotype associated with increase of the protection (CSF38rs22273228CC, CCR78 rs115746638GA, NOS2A8rs169498CT and NOS2A82778GA). The association of the clinical forms paucibacilary and multibacilary with markers showed that to the markers NOS2A8 2778GG, CCR78rs115746638AA and CSF38rs22273228GG there were a strong influence to migration to multibacilary pole and to marker NOS2A8rs169498TT the high proportion was found to the paucibacilary form. Conclusions: Changes in the genes NOS2A, CCR7 and CSF3 can influence the immune response against Mycobacterium leprae. The combination among these polymorphisms alters the risk probability to develop leprosy. The markers type SNP associated to development of the leprosy also are linked to clinical forms and its severity being the polymorphism NOS2A8rs169498TT associated with paucibacilar form and the polymorphisms NOS2A82778GG, CCR78rs115746638AA and CSF38rs22273228GG associated to multibacilar form

Purificação, caracterização e análise da atividade bioinseticida, de um inibidor de tripsina em sementes de catanduva (Piptadenia moniliformis)

One Kunitz-type trypsin inhibitors (PmTI) was purified from Piptadenia moniliformis seeds, a tree of the sub-family Mimosoideae, by TCA precipitation, affinity chromatography on immobilized trypsin-Sepharose, DEAE cellulose (ion exchange) and Superose 12 (molecular exclusion) column FPLC/AKTA. The inhibitor has Mr of 25 kDa by SDS-PAGE and chromatography molecular exclusion. The N-terminal sequence of this inhibitor showed high homology with other family Kunitz inhibitors. This also stable variations in temperature and pH and showed a small decrease in its activity when incubated with DDT in the concentration of 100mM for 120 minutes. The inhibition of trypsin by PmTI was competitive, with Ki of 1.57 x10-11 M. The activity of trypsin was effectively inhibited by percentage of inhibition of 100%, among enzymes tested, was not detected inhibition for the bromelain, was weak inhibitor of pancreatic elastase (3.17% of inhibition) and inhibited by 76.42% elastase of neutrophils, and inhibited in a moderate, chymotrypsin and papain with percentage of inhibition of 42.96% and 23.10% respectively. In vitro assays against digestive proteinases from Lepidoptera, Diptera and Coleoptera pests were carried out. Several degrees of inhibition were found. For Anthonomus grandis and Ceratitis capitata the inhibition was 89.93% and 70.52%, respectively, and the enzymes of Zabrotes subfasciatus and Callosobruchus maculatus were inhibited by 5.96% and 9.41%, respectively, and the enzymes of Plodia. interpunctella and Castnia licus were inhibited by 59.94% and 23.67, respectively. In vivo assays, was observed reduction in the development of larvae in 4rd instar of C. capitata, when PmTI was added to the artificial diet, getting WD50 and LD50 of 0.30% and 0.33%, respectively. These results suggest that this inhibitor could be a strong candidate to plant management programs cross transgenic

Papel da proteína de reparo XPC na regulação das proteínas de reparo APE1, OGG1 e PARP-1 em células humanas e de camundongos

studies using UV as a source of DNA damage. However, even though unrepaired UV-induced DNA damages are related to mutagenesis, cell death and tumorigenesis, they do not explain phenotypes such as neurodegeneration and internal tumors observed in patients with syndromes like Xeroderma Pigmentosum (XP) and Cockayne Syndrome (CS) that are associated with NER deficiency. Recent evidences point to a role of NER in the repair of 8-oxodG, a typical substrate of Base Excision Repair (BER). Since deficiencies in BER result in genomic instability, neurodegenerative diseases and cancer, it was investigated in this research the impact of XPC deficiency on BER functions in human cells. It was analyzed both the expression and the cellular localization of APE1, OGG1 e PARP-1, the mainly BER enzymes, in different NER-deficient human fibroblasts. The endogenous levels of these enzymes are reduced in XPC deficient cells. Surprisingly, XP-C fibroblasts were more resistant to oxidative agents than the other NER deficient fibroblasts, despite presenting the highest of 8-oxodG. Furthermore, subtle changes in the nuclear and mitochondrial localization of APE1 were detected in XP-C fibroblasts. To confirm the impact of XPC deficiency in the regulation of APE1 and OGG1 expression and activity, we constructed a XPC-complemented cell line. Although the XPC complementation was only partial, we found that XPC-complemented cells presented increased levels of OGG1 than XPC-deficient cells. The extracts from XPC-complemented cells also presented an elevated OGG1 enzimatic activity. However, it was not observed changes in APE1 expression and activity in the XPCcomplemented cells. In addition, we found that full-length APE1 (37 kDa) and OGG1- α are in the mitochondria of XPC-deficient fibroblasts and XPC-complemented fibroblasts before and after induction of oxidative stress. On the other hand, the expression of APE1 and PARP-1 are not altered in brain and liver of XPC knockout mice. However, XPC deficiency changed the APE1 localization in hypoccampus and hypothalamus. We also observed a physical interaction between XPC and APE1 proteins in human cells. In conclusion, the data suggest that XPC protein has a role in the regulation of OGG1 expression and activity in human cells and is involved mainly in the regulation of APE1 localization in mice. Aditionally, the response of NER deficient cells under oxidative stress may not be only associated to the NER deficiency per se, but it may include the new functions of NER enzymes in regulation of expression and cell localization of BER proteins

Co-infecção pelo vírus da hepatite G em pacientes infectados pelo Leihsmania chagasi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Modelagem molecular na caracterização eletrônica de oligopeptídeos e na descrição quântica da interação fármaco-receptor

In this dissertation, the theoretical principles governing the molecular modeling were applied for electronic characterization of oligopeptide α3 and its variants (5Q, 7Q)-α3, as well as in the quantum description of the interaction of the aminoglycoside hygromycin B and the 30S subunit of bacterial ribosome. In the first study, the linear and neutral dipeptides which make up the mentioned oligopeptides were modeled and then optimized for a structure of lower potential energy and appropriate dihedral angles. In this case, three subsequent geometric optimization processes, based on classical Newtonian theory, the semi-empirical and density functional theory (DFT), explore the energy landscape of each dipeptide during the search of ideal minimum energy structures. Finally, great conformers were described about its electrostatic potential, ionization energy (amino acids), and frontier molecular orbitals and hopping term. From the hopping terms described in this study, it was possible in subsequent studies to characterize the charge transport propertie of these peptides models. It envisioned a new biosensor technology capable of diagnosing amyloid diseases, related to an accumulation of misshapen proteins, based on the conductivity displayed by proteins of the patient. In a second step of this dissertation, a study carried out by quantum molecular modeling of the interaction energy of an antibiotic ribosomal aminoglicosídico on your receiver. It is known that the hygromycin B (hygB) is an aminoglycoside antibiotic that affects ribosomal translocation by direct interaction with the small subunit of the bacterial ribosome (30S), specifically with nucleotides in helix 44 of the 16S ribosomal RNA (16S rRNA). Due to strong electrostatic character of this connection, it was proposed an energetic investigation of the binding mechanism of this complex using different values of dielectric constants (ε = 0, 4, 10, 20 and 40), which have been widely used to study the electrostatic properties of biomolecules. For this, increasing radii centered on the hygB centroid were measured from the 30S-hygB crystal structure (1HNZ.pdb), and only the individual interaction energy of each enclosed nucleotide was determined for quantum calculations using molecular fractionation with conjugate caps (MFCC) strategy. It was noticed that the dielectric constants underestimated the energies of individual interactions, allowing the convergence state is achieved quickly. But only for ε = 40, the total binding energy of drug-receptor interaction is stabilized at r = 18A, which provided an appropriate binding pocket because it encompassed the main residues that interact more strongly with the hygB - C1403, C1404, G1405, A1493, G1494, U1495, U1498 and C1496. Thus, the dielectric constant ≈ 40 is ideal for the treatment of systems with many electrical charges. By comparing the individual binding energies of 16S rRNA nucleotides with the experimental tests that determine the minimum inhibitory concentration (MIC) of hygB, it is believed that those residues with high binding values generated bacterial resistance to the drug when mutated. With the same reasoning, since those with low interaction energy do not influence effectively the affinity of the hygB in its binding site, there is no loss of effectiveness if they were replaced.

Antígenos da forma amastigota de Leishmania chagasi identificados por dupla varredura da biblioteca de cDNA

Control of human visceral leishmaniasis in endemic regions is hampered in part by the lack of knowledge with respect of the role reservoirs and vector. In addition, there is not yet an understanding of how non-symptomatic subclinical infection might influence the maintenance of infection in a particular locality. Of worrisome is the limited accessibility to medical care in places with emerging drug resistance. There is still no available protective vaccine either for humans or other reservoirs. Leishmania species are protozoa that express multiple antigens which are recognized by the vertebrate immune system. Since there is not one immunodominant epitope recognized by most hosts, strategies must be developed to optimize selection of antigens for prevention and immunodiagnosis. For this reason, we generated a cDNA library from the intracellular amastigote form of Leishmania chagasi, the causative agent of South American visceral leishmaniasis. We employed a two-step expression screen of the library to systematically identify T and T-dependent B cell antigens. The first step was aimed at identifying the largest possible number of clones producing an epitope-containing polypeptide with a pool of sera from Brazilians with documented visceral leishmaniasis. After removal of clones encoding heat shock proteins, positive clones underwent a second step screen for their ability to cause proliferation and IFN-γ responses of T cells from immune mice. Six unique clones were selected from the second screen for further analysis. The clones encoded part of the coding sequence of glutamine synthetase, transitional endoplasmic reticulum ATPase, elongation factor 1γ, kinesin K-39, repetitive protein A2, and a hypothetical conserved protein. Humans naturally infected with L. chagasi mounted both cellular and antibody responses to these protein Preparations containing multiple antigens may be optimal for immunodiagnosis and protective vaccines against Leishmania

Efetividade do uso tópico de fluoreto e da escovação no controle de cáries produzidas "in vivo"

The objective of this clinical study was to evaluate the effectiveness of the toothbrushing with and without fluoride and the daily fluoride rinse (NaF 0.05%) on produced white spot, in vivo. This was a clinical study, controlled, randomized and triple blind. Thirty patients were selected for orthodontics reasons from Orthodontics Specialization Course at the Brazilian Dental Association - Section of Rio Grande do Norte. In this study it was used 4 bicuspid upper and lower. They had orthodontic reason for extractions, in 35 days, at least. The sample had one hundred and twenty teeth that received orthodontic bands. The bands were fixed with polycarboxylate cement, and there was a space standardized between bands and one surface of teeth. The four bicuspid of each patients were randomized and nominated as A, B, C and D. These nominations determinated the sequence of the extractions and what was done in each tooth. All the patients had been submitted to the toothbrushing with or without fluoride for 35 days. After this period, the A tooth of each patient was extracted to serve as control. The others teeth (B, C and D) were extracted one by each week. The entire sample was analyzed through the clinical examination and by laser fluorescence (DIAGNOdent®) in three different times: before orthodontic bands, 28 days after fixed and then removed the bands and, the last one, 07 days after one of the three treatments (toothbrushing with or without fluoride, tooth paste with fluoride and mouth rinse with fluoride). At the beginning all groups (A, B, C and D) had the same conditions, no significant difference was found. The same situation was found in a clinical examination. The results of the DIAGNOdent® for the groups that used tooth paste without fluoride, with fluoride and mouth rinse with fluoride, after 28 days, there was no significant difference. Clinically, the white spot was formed in all teeth after 28 days. When it was compared the three treated groups, the group without fluoride in tooth paste had worst result than the others groups. But there was no significant association between the number of active and inactive white spots and the type of treatment that the teeth had received. The demineralization of the enamel surface, under the orthodontic bands, it happened in a few weeks. The exposition of the white spots in oral environmental resulted in an improvement, but it was not enough to return to the values from the base line, either for the toothbrushing and/or the use of fluorite mouth rinse. Mouth rinse and toothpaste with fluoride have showed to reduce the incidence of demineralization in the enamel, but none seems to be superior to another one in an in vivo study

Plant or fungal sequences? An alternative optimized PCR protocol to avoid ITS (nrDNA) misamplification

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morfologia e avaliação do crescimento inicial de plântulas de Bactris gasipaes Kunth. (arecaceae) em diferentes substratos

Este trabalho teve por objetivos descrever a morfologia dos diásporos, as fases da germinação e determinar o substrato mais adequado para o crescimento inicial de plântulas de pupunha. Periodicamente, unidades representativas de cada fase de germinação foram retiradas para a descrição da seqüência dos eventos morfológicos. Os substratos usados para o crescimento inicial foram Plantmax HT, areia, terra (latossolo roxo) e outro com proporções iguais de terra, areia e esterco (TAE). Avaliou-se o crescimento inicial das plântulas aos 101 dias após o transplante, com base na sua altura, número de folhas, comprimento e largura das folhas. Observou-se que as sementes são albuminosas, com endosperma oleaginoso e de consistência relativamente dura. O embrião é lateral, periférico e relativamente indiferenciado, de forma cônica. A germinação inicia-se com o desenvolvimento de uma massa de células indiferenciadas na depressão micropilar. Posteriormente, esta massa de células torna-se cilíndrica, com a diferenciação dos primórdios caulinar e radicular. O primórdio caulinar é constituído por três bainhas envolvendo a primeira folha. Estas se abrem sucessivamente, permitindo a emergência da folha primária. Entre os substratos testados, de acordo com os parâmetros avaliados, os mais adequados para crescimento inicial de mudas de Bactris gasipaes Kunth foram o TAE e o Plantmax.

Mobilidade do sulfentrazone em Nitossolo Vermelho e em Neossolo Quartzarênico

O objetivo deste trabalho foi determinar a mobilidade do herbicida sulfentrazone em duas classes de solos (Nitossolo Vermelho e Neossolo Quartzarênico), em função de índices pluviométricos crescentes, sendo, portanto, influenciado pelas propriedades químicas e físicas dos solos com diferentes teores de ferro. em tubos de PVC de 10 cm de diâmetro por 50 cm de comprimento, preenchidos com os solos e saturados com água a 65% (p/p), foi aplicado o sulfentrazone (800 g ha-1). Na seqüência, foram simuladas chuvas diárias de 10 mm até atingir os índices pluviométricos de 30, 60 e 90 mm. Os tubos de PVC foram então desmontados - semeando-se Sorghum bicolor nas profundidades correspondentes de 2,5; 7,5; 12,5; 17,5; 22,5; e 30,0 cm da superfície do tubo - e mantidos em casa de vegetação por 15 dias, para avaliação da germinação e do crescimento inicial das plântulas. No final do experimento foram avaliadas as alterações morfofisiológicas que caracterizavam os efeitos tóxicos do produto, além de se medir o comprimento da parte aérea até a última lígula visível e a fitomassa seca das plantas. Quando sob precipitação de 90 mm no Neossolo Quartzarênico, o sulfentrazone formou uma banda de arraste de até 12,5 cm e, no Nitossolo Vermelho, até os 7,5 cm.

Mobilidade do sulfentrazone em latossolo vermelho e em chernossolo

Este trabalho objetivou determinar a mobilidade do sulfentrazone em duas classes de solos em função de índices pluviométricos, bem como possíveis influências das propriedades químicas e físicas de Latossolo Vermelho e Chernossolo com diferentes teores de ferro na ação do herbicida. Foram utilizados como recipientes 36 tubos de PVC de 10 cm de diâmetro por 50 cm de comprimento. Os recipientes foram preenchidos com os solos e umedecidos a 65% (p/p) da capacidade de saturação, quando se fez a aplicação do sulfentrazone (800 g ha-1 de i.a.) na área exposta dos solos. Na seqüência, foram simuladas chuvas diárias de 10 mm até atingir o índice pluviométrico desejado (30, 60 e 90 mm), sendo, posteriormente, desmontados seis tubos de cada solo (com e sem aplicação). Foram semeadas cinco sementes de sorgo (Sorghum bicolor) nas profundidades de 2,5; 7,5; 12,5; 17,5; 22,5; e 30,0 cm, mantidas em casa de vegetação por 15 dias para avaliação da germinação e do crescimento inicial. Decorrido esse tempo, foi realizada avaliação de possíveis alterações morfofisiológicas que pudessem ser caracterizadas como efeitos tóxicos do produto e mediu-se o comprimento da parte aérea até a última lígula visível. As partes aéreas foram secas em estufa com circulação forçada de ar (70 ºC por 96 horas), para obtenção de matéria seca. No Chernossolo ocorreu uniformidade da distribuição do produto ao longo do tubo, proporcional à precipitação, e no Latossolo Vermelho o sulfentrazone foi pouco móvel, permanecendo na camada superficial, independentemente da precipitação.

Licenciamento ambiental em postos de revenda de combustíveis à luz dos princípios constitucionais de defesas do meio ambiente: o caso do município de Natal/RN

This study approaches the question of the administrative procedure of the environmental licensing of ranks of fuel resale, taking as basis a study case of the reality of the City of Natal/RN (Brazil). For in such a way, it was done a retrospect on the evolution of the system of road transport in Brazil, having started to briefly analyze the urbanization process that if after accented in the capitals of the Brazilian States the decade of 1950, relating these subjects with the growth of the sector of fuel resale. After that, it was transferred boarding of the defense of the national environment to the light of ambient principles constitutional. In the sequence, a boarding on the ambient guardianship in the Federal Constitution of 1988 was made, treating basically specifies and on the national urban politics and the national politics of the environment, with its instruments. In the sequence, it was transferred the analysis of the abilities and attributions of the National Advice of the Environment (CONAMA) and its Resolutions, for then only enter in the most important part of this work: an analysis of the environmental licensing of ranks of fuel resale, in the reality of the City of Natal/RN. Before this specific boarding, it was proceeded specifically a survey and communication from the applicable norms to such establishments (Resolutions of the CONAMA and norms of the ABNT), for after that carrying through an geo-ambient characterization of the City of Natal/RN. Finally, a reflection was made on the possibility of magnifying of the state activity, in terms of guarantees for the responsible members for the environmental policy and of administrative efficiency, through the idea of the ambient regulation. For the accomplishment of this study, it was proceeded research in diverse sources such as books, magazines, sites of the Internet, periodicals, thesis and dissertations, among others material, beyond visits the agencies that direct or indirectly act with the ambient defense and as fuel resale, such as Secretariat of Environment and Urbanism of the City of Natal (SEMURB), Institute of Economic Development and Environment of RN (IDEMA), Brazilian Institute of the Environment and the Natural resources You renewed, Executive Management of RN (IBAMA/RN), Public prosecutor's office of the Environment of Natal (Public prosecution service of the RN), National Agency of Oil (ANP) and Union of the Retailing of Derivatives of Oil of the RN, among others. To the end, satiated regulation is observed that although on the substance of the ambient licensing in ranks of fuel resale, also with federal, state and municipal norms, the municipal Public Power is very far from the fulfilment of its institutional functions, in the question environmental policy of these establishments, a time that few are the permitted ranks of resale in the city of Natal/RN