Cytogenetic and random amplified polymorphic DNA analysis of Leptodactylus species from rural and urban environments (Anura, Amphibia)

Cytogenetic and random amplified polymorphic DNA analyses carried out in the species Leptodactylus podicipinus, L. ocellatus, L. labyrinthicus, and L. fuscus from rural and urban habitats of the northwest region of São Paulo State, Brazil, showed that the karyotypes (2n = 22), constitutive heterochromatin distribution and nucleolus organizer region (NOR) location did not differ between the populations from the two environments. The in situ hybridization with an rDNA probe confirmed the location of the NORs on chromosome 8 revealing an in tandem duplication of that region in one of the chromosomes of L. fuscus. DAPI showed that part of the C-band-positive heterochromatin is rich in AT, including that in the proximity the NORs in L. podicipinus and L. ocellatus. The molecular analyses showed that the two populations (urban and rural) of L. podicipinus and L. fuscus are similar from a genetic point of view. The urban and rural populations of species L. ocellatus and L. labyrinthicus showed differences in genetic structures, probably due to urbanization which interferes with the dispersion of those frogs. The marked differences observed between the two populations of L. ocellatus can be representing the cryptic condition of the species. Unweighted pair-group method of analysis and genetic distance analysis detected the genetic proximity between L. ocellatus and L. fuscus. The results indicate that there was no reduction in the genetic diversity in the populations from the urban environment; however, the survival of these frogs would not be guaranteed in the case of an increase in human impact especially for populations of L. labyrinthicus and L. ocellatus. ©FUNPEC-RP.

Molecular characterization of Pleurotus ostreatus commercial strains by random amplified polymorphic DNA (RAPD)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Haplotype frequencies based on eight polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from two different geographical regions of Brazil

The Brazilian population represents an admixture of native Amerindians, Portuguese settlers and Africans who were brought as slaves during the colonization period that began in the 16th century and was followed by waves of immigrations of Europeans and Asians in the 20th century. The contribution of these different ethnic groups to the constitution of Brazilian populations from different geographic regions is variable and, in addition to environmental factors, might act by determining different allele profiles among Brazilian populations from different regions. We studied polymorphic sites at the 3' untranslated region of the HLA-G gene in individuals from a Northeastern Brazilian region and compared them to our previously published data about a Southeastern Brazilian region, located at a distance of 2589 km. Our results showed that most polymorphic sites present a similar distribution in both populations, except for the lower frequency of the +3003C allele in the Northeastern population compared to the Southeastern population. Although differences in genotypic distribution were only significant for the +3003 locus (P = 0.0201), the diversity of haplotypes was distinct for each population. These results are important for casecontrol studies on the association of human leucocyte antigen-G polymorphism with disease and also in terms of the genetic structure of two distinct Brazilian populations.

Polymorphic variants of the multidrug resistance gene Mdr1a and response to antiepileptic drug treatment in the kindling model of epilepsy

Allelic variants of the human P-glycoprotein encoding gene MDR1 (ABCB1) are discussed to be associated with different clinical conditions including pharmacoresistance of epilepsy. However, conflicting data have been reported with regard to the functional relevance of MDR1 allelic variants for the response to antiepileptic drugs. To our knowledge, it is not known whether functionally relevant genetic polymorphisms also occur in the two genes (Mdr1a/Abcb1a, Mdr1b/Abcb1b) coding for P-glycoprotein in the brain of rodents. Therefore, we have started to search for polymorphisms in the Mdr1a gene, which governs the expression of P-glycoprotein in brain capillary endothelial cells in rats. In the kindling model of temporal lobe epilepsy, subgroups of phenytoin-sensitive and phenytoin-resistant rats were selected in repeated drug trials. Sequencing of the Mdr1a gene coding sequence in the subgroups revealed no general differences between drug-resistant and drug-sensitive rats of the Wistar outbred strain. A comparison between different inbred and outbred rat strains also gave no evidence for polymorphisms in the Mdr1a coding sequence. However, in exon-flanking intron sequences, four genetic variants were identified by comparison between these rats strains. In conclusion, the finding that Wistar rats vary in their response to phenytoin, while having the same genetic background, argues against a major impact of Mdr1a genetics on pharmacosensitivity to antiepileptic drugs in the amygdala kindling model.

Metabolism, oxidative stress and territorial behaviour in a female colour polymorphic cichlid fish

Intrasexual selection on body coloration is thought to play an important role in the evolution of colour polymorphism, but its physiological underpinnings have received limited attention. In the colour polymorphic cichlid Neochromis omnicaeruleus, three fully sympatric female colour morphs— a plain morph (P) and two conspicuously coloured blotched morphs, black-and-white blotched (WB) and orange blotched (OB)—differ in agonistic behaviour. We compared routine metabolic rate (when females were housed in social isolation), short-term energetic costs of interacting with a same-colour rival housed in an adjacent transparent chamber and oxidative stress between the three female colour morphs. WB females had a lower routine metabolic rate compared with the other colour morphs. WB females also had a lower active metabolic rate during inter-female interactions than OB females, while OB females used more oxygen per unit aggressive act than the other two colour morphs. However, there were no consistent differences in oxidative stress between the three morphs. Concerted divergence in colour, behaviour and metabolism might contribute to the evolution of these polymorphisms in sympatry.

Clinical relevance of polymorphic DNA copy number variation (CNV) in African American women with stage I-II breast cancer

Objectives. The chief goal of this study was to analyze copy number variation (CNV) in breast cancer tumors from 25 African American women with early stage breast cancer (BC) using molecular inversion probes (MIP) in order to: (1) compare the degree of CNV in tumors compared to normal lymph nodes, and (2) determine whether gains and/or losses of genes in specific chromosomes differ between pathologic subtypes of breast cancer defined by known prognostic markers, (3) determine whether gains/losses in CN are associated with known oncogenes or tumor suppressor genes, and (4) determine whether increased gains/losses in CN for specific chromosomes were associated with differences in breast cancer recurrence. ^ Methods. Twenty to 37 nanograms of DNA extracted from 25 formalin-fixed paraffin embedded (FFPE) tumor samples and matched normal lymph nodes were added to individual tubes. Oligonucleotide probes with recognition sequences at each terminus were hybridized with a genomic target sequence to form a circular structure. Probes are released from genomic DNA obtained from FFPE samples, and those which have been correctly "circularized" in the proper allele/nucleotide reaction combination are amplified using polymerase chain reaction (PCR) primers. Amplicons were fluorescently labeled and the tag sequences released from the genome homology regions by treatment with uracil-N-glycosylase to cleave the probe at the site where uracils are present, and detected using a complementary tag array developed by Affymetrix. ^ Results. Analysis of CN gains and losses from tumors and normal tissues showed marked differences in tumors with numerous chromosomes affected. Similar changes were not observed in normal lymph nodes. When tumors were stratified into four groups based on expression or lack of expression of the estrogen receptor and HER2/neu, distinct patterns of CNV for different chromosomes were observed. Gains or losses in CN for specific chromosomes correlated with amplifications/deletions of particular oncogenes or tumor suppressor genes (i.e. such as found on chromosome 17) known to be associated with aggressive tumor phenotype and poor prognosis. There was a trend for increases in CN observed for chromosome 17 to correlate inversely with time to recurrence of BC (p=0.14 for trend). CNV was also observed for chromosomes 5, 8, 10, 11, and 16, which are known sites for several breast cancer susceptibility alleles. ^ Conclusions. This study is the first to validate the MIP technique, to correlate differences in gene expression with known prognostic tumor markers, and to correlate significant increases/decreases in CN with known tumor markers associated with prognosis. The results of this study may have far reaching public health implications towards identifying new high-risk groups based on genomic differences in CNP, both with respect to prognosis and response to therapy, and to eventually identify new therapeutic targets for prevention and treatment of this disease. ^

All males are not created equal: Fertility differences depend on gamete recognition polymorphisms in sea urchins

Behaviors, morphologies, and genetic loci directly involved in reproduction have been increasingly shown to be polymorphic within populations. Explaining how such variants are maintained by selection is crucial to understanding the genetic basis of fertility differences, but direct tests of how alleles at reproductive loci affect fertility are rare. In the sea urchin genus Echinometra, the protein bindin mediates sperm attachment to eggs, evolves quickly, and is polymorphic within species. Eggs exposed to experimental sperm mixtures show strong discrimination on the basis of the males’ bindin genotype. Different females produce eggs that nonrandomly select sperm from different males, showing that variable egg–sperm interactions determine fertility. Eggs select sperm with a bindin genotype similar to their own, suggesting strong linkage between female choice and male trait loci. These experiments demonstrate that alleles at a single locus can have a strong effect on fertilization and that reproductive loci may retain functional polymorphisms through epistatic interactions between male and female traits. They also suggest that positive selection at gamete recognition loci like bindin involves strong selection within species on mate choice interactions.

PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori

Genes that are characteristic of only certain strains of a bacterial species can be of great biologic interest. Here we describe a PCR-based subtractive hybridization method for efficiently detecting such DNAs and apply it to the gastric pathogen Helicobacter pylori. Eighteen DNAs specific to a monkey-colonizing strain (J166) were obtained by subtractive hybridization against an unrelated strain whose genome has been fully sequenced (26695). Seven J166-specific clones had no DNA sequence match to the 26695 genome, and 11 other clones were mixed, with adjacent patches that did and did not match any sequences in 26695. At the protein level, seven clones had homology to putative DNA restriction-modification enzymes, and two had homology to putative metabolic enzymes. Nine others had no database match with proteins of assigned function. PCR tests of 13 unrelated H. pylori strains by using primers specific for 12 subtracted clones and complementary Southern blot hybridizations indicated that these DNAs are highly polymorphic in the H. pylori population, with each strain yielding a different pattern of gene-specific PCR amplification. The search for polymorphic DNAs, as described here, should help identify previously unknown virulence genes in pathogens and provide new insights into microbial genetic diversity and evolution.

Computing the structural and vibrational properties of polymorphic organic molecular crystals through van der Waals corrected density functional theory and the electronic properties of organic thin films through microelectrostatic calculations.

The present Thesis reports on the various research projects to which I have contributed during my PhD period, working with several research groups, and whose results have been communicated in a number of scientific publications. The main focus of my research activity was to learn, test, exploit and extend the recently developed vdW-DFT (van der Waals corrected Density Functional Theory) methods for computing the structural, vibrational and electronic properties of ordered molecular crystals from first principles. A secondary, and more recent, research activity has been the analysis with microelectrostatic methods of Molecular Dynamics (MD) simulations of disordered molecular systems. While only very unreliable methods based on empirical models were practically usable until a few years ago, accurate calculations of the crystal energy are now possible, thanks to very fast modern computers and to the excellent performance of the best vdW-DFT methods. Accurate energies are particularly important for describing organic molecular solids, since they often exhibit several alternative crystal structures (polymorphs), with very different packing arrangements but very small energy differences. Standard DFT methods do not describe the long-range electron correlations which give rise to the vdW interactions. Although weak, these interactions are extremely sensitive to the packing arrangement, and neglecting them used to be a problem. The calculations of reliable crystal structures and vibrational frequencies has been made possible only recently, thanks to development of some good representations of the vdW contribution to the energy (known as “vdW corrections”).

Membrane Lipid Profile Monitored By Mass Spectrometry Detected Differences Between Fresh And Vitrified In Vitro-produced Bovine Embryos.

Summary This study aimed to evaluate the impact of vitrification on membrane lipid profile obtained by mass spectrometry (MS) of in vitro-produced bovine embryos. Matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) has been used to obtain individual embryo membrane lipid profiles. Due to conditions of analysis, mainly membrane lipids, most favorably phosphatidylcholines (PCs) and sphingomyelins (SMs) have been detected. The following ions described by their mass-to-charge ratio (m/z) and respective attribution presented increased relative abundance (1.2-20×) in the vitrified group: 703.5 [SM (16:0) + H]+; 722.5 [PC (40:3) + Na]+; 758.5 [PC (34:2) + H]+; 762.5 [PC (34:0) + H]+; 790.5 [PC (36:0) + H]+ and 810.5 [PC (38:4) + H]+ and/or [PC (36:1) + Na]+. The ion with a m/z 744.5 [PCp (34:1) and/or PCe (34:2)] was 3.4-fold more abundant in the fresh group. Interestingly, ions with m/z 722.5 or 744.5 indicate the presence of lipid species, which are more resistant to enzymatic degradation as they contain fatty acyl residues linked through ether type bonds (alkyl ether or plasmalogens, indicated by the lowercase 'e' and 'p', respectively) to the glycerol structure. The results indicate that cryopreservation impacts the membrane lipid profile, and that these alterations can be properly monitored by MALDI-MS. Membrane lipids can therefore be evaluated by MALDI-MS to monitor the effect of cryopreservation on membrane lipids, and to investigate changes in lipid profile that may reflect the metabolic response to the cryopreservation stress or changes in the environmental conditions.

The Use Of Medication And Associated Factors Among Adults Living In Campinas, São Paulo, Brazil: Differences Between Men And Women.

The objective of this study was to verify factors associated with the use of medication by adults, with emphasis on the differences between men and women. It was a population-based, cross-sectional study with cluster sampling conducted in two stages in Campinas in the state of São Paulo in 2008. Among the 2,413 individuals aged 20 or older, the prevalence of use of at least one drug in the three days before the research was 45.4% (95% CI: 41.3 - 49.4) in men and 64.6% (95% CI: 59.8 - 69.2) in women. For adult men over 40 years old who were not working, former smokers, with one or more chronic diseases, with two or more health problems and who sought health care or a health professional in the two weeks preceding the research showed higher prevalence of medication use. Among women, a higher prevalence of use was observed in females over 40, obese, former smokers, who reported a short sleep pattern, with one or more chronic diseases and two or more health problems, and who reported seeking a health care service or professional in the past 15 days. The findings showed some differences in the determinants of drug use in relation to gender, revealing the greater importance of health-related behavior among women.

Microsatellite Markers For Urochloa Humidicola (poaceae) And Their Transferability To Other Urochloa Species.

Urochloa humidicola is a warm-season grass commonly used as forage in the tropics and is recognized for its tolerance to seasonal flooding. This grass is an important forage species for the Cerrado and Amazon regions of Brazil. U. humidicola is a polyploid species with variable ploidy (6X-9X) and facultative apomixis with high phenotypic plasticity. However, this apomixis and ploidy, as well as the limited knowledge of the genetic basis of the germplasm collection, have constrained genetic breeding activities, yet microsatellite markers may enable a better understanding of the species' genetic composition. This study aimed to develop and characterize new polymorphic microsatellite molecular markers in U. humidicola and to evaluate their transferability to other Urochloa species. A set of microsatellite markers for U. humidicola was identified from two new enriched genomic DNA libraries: the first library was constructed from a single sexual genotype and the second from a pool of eight apomictic genotypes selected on the basis of previous results. Of the 114 loci developed, 72 primer pairs presented a good amplification product, and 64 were polymorphic among the 34 genotypes tested. The number of bands per simple sequence repeat (SSR) locus ranged from 1 to 29, with a mean of 9.6 bands per locus. The mean polymorphism information content (PIC) of all loci was 0.77, and the mean discrimination power (DP) was 0.87. STRUCTURE analysis revealed differences among U. humidicola accessions, hybrids, and other Urochloa accessions. The transferability of these microsatellites was evaluated in four species of the genus, U. brizantha, U. decumbens, U. ruziziensis, and U. dictyoneura, and the percentage of transferability ranged from 58.33% to 69.44% depending on the species. This work reports new polymorphic microsatellite markers for U. humidicola that can be used for breeding programs of this and other Urochloa species, including genetic linkage mapping, quantitative trait loci identification, and marker-assisted selection.

Differences in responses to the Oral Health Impact Profile (OHIP14) used as a questionnaire or in an interview

The objective of this study was to compare the completion rates and performance of the Brazilian version of the Oral Health Impact Profile (OHIP14) when applied as an interview or in its original self-reported form. A convenience sample of 74 adult patients was selected in a Dental Clinic (University of Araras, Brazil). One examiner administered the instrument in both formats to participants with an interval of 2 weeks between each administration. Data about dental health condition and socioeconomic status were collected and associated with total OHIP14 scores in both formats using linear regression analyses. No differences were found in the total scores and in each subscale of the OHIP14 according to the form of administration. Higher values of completion were found in the interview format. More severe impacts were recorded in the interview format than in the questionnaire format. Higher values of total OHIP-14 scores in both formats were related to the presence of dental caries. Total OHIP14 scores were not influenced by the method of administration. However, the use of the OHIP14 in the questionnaire format may result in lower completion rates and loss of data.

Weekend/weekday differences in concentrations of ozone, nox, and non-methane hydrocarbon in the metropolitan area of São Paulo

Ozone and inhalable particulate matter are the major air pollutants in the Metropolitan Area of São Paulo, Brazil, a region that has more than 19 million inhabitants and approximately 7 million registered vehicles. Proximity of roadways, adjacent land use, and local circulation are just some of the factors that can affect the results of monitoring of pollutant concentrations. The so-called weekend effect (higher ozone concentrations on weekends than on weekdays) might be related to the fact that concentrations of ozone precursors, such as nitrogen oxides (NOx) and Non Methane-Hydrocarbon (NMHC), are relatively lower on weekends. This phenomenon has been reported in some areas of the United States since the 1970s. The differences between the concentrations of ozone in period of weekend and weekday, were obtained from analysis of data hourly average of CETESB for 2004, studied the precursors to the formation of troposphere ozone, the meteorological variables and traffic profile for RMSP. Because of the proximity to sources of emissions from the station Pinheiros showed higher concentrations of NO and NO² and greater variations to the periods weekend and weekday. With fewer vehicles circulating during the weekend, and consequently less emission of pollutants, it has cleaner air and less concentration of NO and NO², there is the ideal setting to the formation of troposphere ozone, despite the lower concentration of NO². The proximity with the source emissions, aided by the increased availability of solar radiation and the presence of ozone precursors, were factors conditions for the occurrence of weekend effect.

Thermal behavior and decomposition kinetics of rifampicin polymorphs under isothermal and non-isothermal conditions

The thermal behavior of two polymorphic forms of rifampicin was studied by DSC and TG/DTG. The thermoanalytical results clearly showed the differences between the two crystalline forms. Polymorph I was the most thermally stable form, the DSC curve showed no fusion for this species and the thermal decomposition process occurred around 245 ºC. The DSC curve of polymorph II showed two consecutive events, an endothermic event (Tpeak = 193.9 ºC) and one exothermic event (Tpeak = 209.4 ºC), due to a melting process followed by recrystallization, which was attributed to the conversion of form II to form I. Isothermal and non-isothermal thermogravimetric methods were used to determine the kinetic parameters of the thermal decomposition process. For non-isothermal experiments, the activation energy (Ea) was derived from the plot of Log β vs 1/T, yielding values for polymorph form I and II of 154 and 123 kJ mol-1, respectively. In the isothermal experiments, the Ea was obtained from the plot of lnt vs 1/T at a constant conversion level. The mean values found for form I and form II were 137 and 144 kJ mol-1, respectively.