Caracterização da resistência do algodoeiro a Ramularia areola e variabilidade molecular do patógeno

This research was conducted with the aim to study the genetic and pathogenic structure of Ramularia areola isolates collected in Brazil and to characterize the resistance response in cotton plants to ramularia spot. The genetic variability of 28 isolates of R. areola was studied using RAPD markers. The pathogenicity evaluation was realized by the inoculation of 6 isolates on cotton varieties Guazuncho-2 (Gossypium hirsutum) and VH8-4602 (Gossypium barbadense). The inheritance of disease resistance was studied using an artificially inoculated population of F2 individuals derived from the intercross of Guazuncho-2 (susceptible variety) end VH8-4602 (resistant variety), and also the parents and F1 individuals. Molecular polymorphism between the G. hisutum varieties DeltaOpal (suscetible) and CNPA CO-11612 (resistant) was estimated by 118 SSR and 24 AFLP markers. The parental genotypes Guazuncho-2 and VH8-4602 were selected for mapping, and then Recombinant Inbred Lines (RIL´s) derived from this crossing were evaluated with SSR 12 markers. The analysis of population structure of R. areola revealed that the three subpopulations were genetically simillar (Gst=0.18), and the isolates from Goiás and Minas Gerais were more similar to each other (0,92). This probability can be related to the relatively high gene flow among the three subpopulations (Nm=2.20). The isolates R. areola 9.1, from Minas Gerais State and 8.1 and 8.3 from Goiás State were the most aggressive ones to the susceptible variety Guazuncho-2. The variety VH8-4602 presented high level of resistance to ramularia spot. No differential interaction was observed between the pathogens and the analyzed varieties, and the resistance was classified as horizontal. The quantification of disease by number of necrotic lesions and number of spores in individual plants of F1 and F2 generations from the crossing between the varieties Guazuncho-2 and VH8-4602 presented continuous distribution, suggesting polygenic resistance. The resistance is probabilly recessive, since necrotic lesions and sporulation were observed on F1 plants. The molecular polymorphism between DeltaOpal e CNPA CO-11612 lineages was low (6%), then would be difficult to accomplish molecular mapping of disease resistance using this intercross. With the genotyping of the RIL s it was verified that 25% of the markers segregated in the proportions proposed by Mendel s Law and 75% of the studied markers presented segregation distortion in favor to the parental G. hirsutum. Both the low genetic variability of the pathogen and the number of resistance genes suggest that durable genetic resitance may be achieved

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.

Associação da frequência alélica dos polimorfismos dos genes do sistema renina angiotensina com a força muscular e pressão arterial de idosas

Physiological changes induced by the aging process is dynamic and progressive, reducing the adaptability and independence of older people and may be influenced by genetic and environmental factors. Thus the aim of this thesis was to investigate the association between polymorphism of the ACE gene ID and the phenotypes of muscular strength and blood pressure of 62 elderly Brazilian (67.35 ± 5.66 years) during a 16-week program of supervised training. The elderly women were stratified by age, with the group 1 (G1, n = 34) <70 years and group 2 (G2 n = 28) ≥ 70 years, and in three groups by ACE, ACE-II (n = 8) ACE- DD (n = 35) and ACE-ID (n = 19). The level of muscle strength was evaluated by the method of maximum repetitions and measures of blood pressure (BP) were measured before and after training (PAPré1 and PAPós1) and before and after each training session (PAPre2 and PAPós2), in place of training. DNA samples were isolated from peripheral blood leukocytes polymorphism and insertion / deletion (ID) of the ACE gene (rs1800795) was genotyped by polymerase chain reaction (PCR) plus PCR-confirmatory. The genotype distribution of the polymorphism ID attended the prerogatives of Hardy-Weitíherg. There was variation in power levels before and after training and the age between groups (t-test) and the ACE polymorphism (ANOVA) (p <0.05). Depending on the results it was concluded that resistance training helps to reduce SBP and increased muscle strength of upper and lower limbs when considering the age and ACE polymorphism. In this study the Elderly carriers of the D allele were more reactive to changes in BP resistance training. This study was multidisciplinary project involving researchers in the areas Medical, Physical Education, Pharmacy, Nutrition, Gerontology and Statistics. This fulfilled the requirements of the multidisciplinary Graduate Program in Health Sciences

Estudo dos genes do complexo do antígeno leucocitário humano (hla) associados à susceptibilidade ao diabetes mellitus tipo 1

Of all of the genes associated with the development of Diabetes mellitus type 1 (T1D), the largest contribution comes from the genes in the Human Leukocyte Antigen (HLA) region, mostly the class II DR e DQ genes. Specific combinations of alleles DRB1, DQA1 and DQB1 constituting haplotypes, and further, a combination of more than one haplotype, providing multilocus genotypes are associated with susceptibility, protection and neutrality to DM1. Thus, the aim of present study was to verified the association of polymorphisms of HLA genes class II with susceptibility to type 1 diabetes mellitus (T1D). Ninety-two patients with T1D and 100 individuals normoglycemics (NG) aged between 6 and 20 years were studied. Genomic DNA was obtained from peripheral whole blood, collected in EDTA tube, using the extraction kit Illustra Triple Prep®, GE Healthcare. For HLA typing was used DNA LABType system by One Lambda kit applying Luminex® technology to the method of PCRSSO typing reverse. The alleles DRB1*03:01, *04:05, *04:01, *04:02, DQA1*03:01g, *05:01g, DQB1*02:01g, *03:02, the haplotypes DRB1*03:01-DQA1*05:01-DQB1*02:01, DRB1*04:05-DQA1*03:01g-DQB1*03:02, DRB1*04:02-DQA1*03:01g-DQB1*03:02, DRB1*04:01-DQA1*03:01g-DQB1*03:02 and DR3-DQ2/DR4-DQ8 genotype were significantly associated with the chance of developing T1D. The alleles DRB1*11:01, *15:03, *15:01, *13:01, DQA1*01:02, *04:01g, *01:03, DQB1*06:02, *03:01g, *06:03, *04:02, the haplotypes DRB1*11:01-DQA1*05:01-DQB1*03:01, DRB1*13:01-DQA1*01:03-DQB1*06:03 and DRX-DQX/DRX-DQX genotype, formed by other than the DR3-DQ2 or DR4-DQ8 haplotypes, were significantly associated with T1D protection Despite the major racial Brazilian, even at the regional level, these results are similar to the majority of alleles, genotypes and haplotypes of HLA class II-related susceptibility or resistance to T1D, extensively described in the literature for Caucasian population. Children with age at diagnosis less than 5 years of age had significantly higher frequency of the heterozygous genotype DR3-DQ2/DR4-DQ8 compared to children with age at diagnosis than 5 years old. These results also demonstrate strong association of the genetic profile of the class II HLA for this age group, possibly associated with the severity and rapid progression to the onset of T1D. The knowledge of HLA class II genes may be useful in genetic screens that allow the prediction of T1D

Prospecção de genes de interesse biotecnológico : uma abordagem metagenômica

The total number of prokaryotic cells on Earth has been estimated at 4 to 6x1030 and only about 1% of microorganisms present in the environment can be cultivated by standard techniques of cultivation and plating. Therefore, it is a huge biological and genetic pool that can be exploited, for the identification and characterization of genes with biotechnological potential. Within this perspective, the metagenomics approach was applied in this work. Functional screening methods were performed aiming to identify new genes related to DNA repair and / or oxidative stress resistance, hydrocarbon degradation and hydrolytic activities (lipase, amylase and protease). Metagenomic libraries were built utilizing DNA extracted from soil samples collected in João Câmara RN. The libraries were analyzed functionally using specific substrate containing solid medium (hydrolytic activity), supplemented with H2O2 (DNA repair and / or resistance to oxidative stress) and liquid medium supplemented with light Arabian oil (activity, degradation of hydrocarbons). After confirmation of activity and exclusion of false-positive results, 49 clones were obtained, being 2 positive for amylase activity, 22 resistant to oxidative stress generated by H2O2 and 25 clones active for hydrocarbons degradation. Analysis of the sequences showed hypothetical proteins, dienelactona hydrolase, DNA polymerase, acetyltransferase, phosphotransferase, methyltransferase, endonucleases, among other proteins. The sequence data obtained matched with the functions tested, highlighting the success of metagenomics approaches combined with functional screening methods, leading to very promising results

Identificação de genes em Chromobacterium violaceum relacionados à resposta ao estresse

The sequencing of the genome of Chromobacterium violaceum identified one single circular chromosome of 4.8 Mb, in which approximately 40% of the founded ORFs are classified as hypothetical conserved or hypothetical. Some genic regions of biotechnological and biological interest had been characterized, e. g., environmental detoxification and DNA repair genes, respectively. Given this fact, the aim of this work was to identify genes of C. violaceum related to stress response, as the ones involved with mechanisms of DNA repair and/or genomic integrity maintenance. For this, a genomic library of C. violaceum was built in Escherichia coli strain DH10B (RecA-), in which clones were tested to UVC resistance, resulting in five candidates clones. In the PLH6A clone were identified four ORFs (CV_3721 to 3724). Two ORFs, CV_3722 and CV_3724, were subcloned and a synergic complementation activity was observed. The occurrence of an operon was confirmed using cDNA from C. violaceum in a RT-PCR assay. Further, it was observed the induction of the operon after the treatment with UVC. Thus, this operon was related to the stress response in C. violaceum. The mutagenesis assay with rifampicin after the treatment with UVC light showed high frequency of mutagenicity for the ORF CV_3722 (Pol III δ subunit). In this way, we propose that the C. violaceum δ subunit can act in DH10B in the translesion synthesis using Pol IV in a RecA independent-manner pathway. In growth curve assays other four clones (PLE1G, PLE7B, PLE10B and PLE12H) were able to complement the function at the dose 5 J/m2 and in mutagenicity assays PLE7B, PLE10B and PLE12H showed frequencies of mutation with significant differences upon the control (DH10B), demonstrating that in some way they are involved with the stress response in C. violaceum. These clones appear to be interrelated, probably regulated by a messenger molecule (eg., nucleotide c-di-GMP) and/or global regulatory molecule (eg., σS subunit of RNA polymerase).The results obtained contribute for a better genetic knowledge of this specie and its response mechanisms to environmental stress.