Programa de Pós-Graduação em Genética e Biologia Molecular
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Master Thesis Caracterização de dois cDNAs homológos e uma AP endonuclease em cana-de-açúcar(Universidade Federal do Rio Grande do Norte, 2009-02-27) Oliveira, Andrea de Lima; Scortecci, Kátia Castanho; http://lattes.cnpq.br/4808910380593455; http://lattes.cnpq.br/9710432901164124; Lima, Lucymara Fassarela Agnez; http://lattes.cnpq.br/1083882171718362; Sluys, Marie-anne Van; http://lattes.cnpq.br/5131787064674647The genome of all organisms is subject to injuries that can be caused by endogenous and environmental factors. If these lesions are not corrected, it can be fixed generating a mutation which can be lethal to the organisms. In order to prevent this, there are different DNA repair mechanisms. These mechanisms are well known in bacteria, yeast, human, but not in plants. Two plant models Oriza sativa and Arabidopsis thaliana had the genome sequenced and due to this some DNA repair genes have been characterized. The aim of this work is to characterized two sugarcane cDNAs that had homology to AP endonuclease: scARP1 and scARP3. In silico has been done with these two sequences and other from plants. It has been observed domain conservation on these sequences, but the cystein at 65 position that is a characteristic from the redox domain in APE1 protein was not so conservated in plants. Phylogenetic relationship showed two branches, one branch with dicots and monocots sequence and the other branch with only monocots sequences. Another approach in order to characterized these two cDNAs was to construct overexpression cassettes (sense and antisense orientation) using the 35S promoter. After that, these cassettes were transferred to the binary vector pPZP211. Furthermore, previously in the laboratory was obtained a plant from nicotiana tabacum containing the overexpression cassette in anti-sense orientation. It has been observed that this plant had a slow development and problems in setting seeds. After some manual crossing, some seeds were obtained (T2) and it was analyzed the T2 segregation. The third approach used in this work was to clone the promoter region from these two cDNAs by PCR walking. The sequences obtained were analyzed using the program PLANTCARE. It was observed in these sequences some motives that may be related to oxidative stress responseMaster Thesis Caracterização dos genes scMUTM1 e scMUTM2 de cana-de-açúcar(Universidade Federal do Rio Grande do Norte, 2007-02-23) Medeiros, Viviane Katielly Silva; Scortecci, Kátia Castanho; ; http://lattes.cnpq.br/4808910380593455; ; http://lattes.cnpq.br/0635169659175207; Lima, Lucymara Fassarela Agnez; ; http://lattes.cnpq.br/1083882171718362; Machado, Carlos Renato; ; http://lattes.cnpq.br/6306925202374274Plants are organisms sessile and because of this they are susceptible to genotoxic effects due to environmental exposure such as light [including ultraviolet (UV)], heat, drought and chemicals agents. Therefore, there are differents pathways in order to detect a lesion and correct. These pathways are not well known in plants. The MutM/Fpg protein is a DNA glycosylase that is responsible for detect and correct oxidative lesions. In the sugarcane genome, it was found two possible cDNAs that had homology to this protein: scMUTM1 and scMUTM2. The aim of this work was to characterize the role of these cDNAs in plants. In order to do this, the expression level after oxidative stress was evaluated by semiquantitative RT-PCR. Another point analyzed in order to obtain the full-length gene, it was to use a sugarcane genomic library that was hybridized with both cDNAs as a probe. It was found two clones that will bought and sequenced. The promoter region was also cloned. It was obtained sequences only for scMUTM2 promoter region. The sequences obtained were divided into six groups. It was found regulatory motifs such as TATA-box, CAAT-box, oxidative stress element response and regulatory regions that response to light. The other point analyzed was to characterize the N-terminal region by PCR constructs. These constructs have deletions at 5 region. These sequences were introduce into Escherichia coli wild type strain (CC104) and double mutant (CC104mutMmutY). The results showed that proteins with deletions of scMUTM1 N-terminal region were able to complement the Fpg and MutY-glycosylase deficiency in CC104 mutMmutY reducing the spontaneous mutation frequencyMaster Thesis Caracterização da resistência do algodoeiro a Ramularia areola e variabilidade molecular do patógeno(Universidade Federal do Rio Grande do Norte, 2007-02-23) Lucena, Valeska Silva; Hoffmann, Lúcia Vieira; ; http://lattes.cnpq.br/1552220402943995; ; http://lattes.cnpq.br/4490260435452349; Suassuna, Nelson Dias; ; http://lattes.cnpq.br/4344026717541727; Giband, Marc; ; http://lattes.cnpq.br/0551184684899446This 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 achievedMaster Thesis Diversificação cariotípica em cinco espécies de moréias do Atlântico Ocidental (Anguilliformes)(Universidade Federal do Rio Grande do Norte, 2007-02-23) Vasconcelos, Antônio Jales Moraes; Molina, Wagner Franco; ; http://lattes.cnpq.br/8437464961129518; ; http://lattes.cnpq.br/8784507023640851; Affonso, Paulo Roberto Antunes de Mello; ; http://lattes.cnpq.br/4149576837587664; Panosso, Renata de Fátima; ; http://lattes.cnpq.br/2543467420133635The Anguiliformes is constituted by 15 families, 141 sorts and 737 species. In this group eight families possess at least one karyotyped species, where a prevalence of karyotypes with 2n=38 is evidenced chromosomes and high NF, apparently basal for the Anguiliformes. The only family who shows a different karyotypic pattern from the others is the Muraenidae family. In this, of the eight species already described, all of them present 2n=42 chromosomes. Despite the dimension of this Order, few species present karyotypics descriptions. In the present work, a species of Ophichthidae, Myrichthys ocellatus (2n=38, 8m+14sm+10st+6a, NF=70) and three species of Muraenidae, Enchelycore nigricans (2n=42, 6m+8sm+12st+16a, NF=68), Gymnothorax miliaris (2n=42, 14m+18sm+10st, NF=84), Gymnothorax vicinus (2n=42, 8m+6sm+28a, NF=56) and Muraena pavonina (2n=42, 6m+4sm+32a, NF=52), collected in the coast of the Rio Grande do Norte state, Saint Peter and Paul Rocks and in the coast of Bahia state were analyzed. Mitotics chromosomes had been gotten through mitotic stimulation with yeasts. Among the analyzed species, it is observed the presence of characteristic large metacentric chromosomic pairs (≅10µm). As for the structural standard, heterochromatics regions in these species in centromeric position of the majority of the chromosomic pairs and simple ribosomal sites had been evidenced. For the Ophichthidae family, the gotten data corroborate the hypothesis of karyotypic diversification mediated by the occurrence of pericentrics inversions and robertsonians rearrangements, while in the Muraenidae, the identification of larger chromosomic values (2n=42), suggests derived karyotypes, possibly caused by possible chromosomic fissionsMaster Thesis Avaliação da genotoxicidade das águas sueprficiais da Barragem Engenheiro Armando Ribeiro Gonçalves, Assu/RN(Universidade Federal do Rio Grande do Norte, 2007-02-28) Cabral, Thiago de Melo; Medeiros, Sílvia Regina Batistuzzo de; Panosso, Renata de Fátima; ; http://lattes.cnpq.br/2543467420133635; ; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8; ; http://lattes.cnpq.br/1599632696789745; Hacon, Sandra de Souza; ; http://lattes.cnpq.br/7653379361147439; Lima, Lucymara Fassarela Agnez; ; http://lattes.cnpq.br/1083882171718362Master Thesis Análise molecular dos éxons 8 a 11 do gene da p53 em amostras de câncer de colo de útero no Rio Grande do Norte(Universidade Federal do Rio Grande do Norte, 2007-02-23) Barbosa, Rodrigo Niskier Ferreira; Meissner, Rosely de Vasconcellos; ; http://lattes.cnpq.br/7518973237001429; ; http://lattes.cnpq.br/7870682050323416; Rabenhorst, Silvia Helena Baren; ; http://lattes.cnpq.br/3868264006150535; Fernandes, José Veríssimo; ; http://lattes.cnpq.br/7078820975978056; Blaha, Carlos Alfredo Galindo; ; http://lattes.cnpq.br/2307806644081146Mutations on TP53 gene are common in human cancer but not in cervical cancer where they are rarely found and the inactivation and degradation of p53 protein are attributed to the action of E6 viral oncogene from high risk human papillomavirus (HPV). Analysis of cervical cancer cell lines suggests that HPV negative samples shows mutation on TP53, but clinical approaches didn t confirmed this hypothesis. However, in most TP53 mutations studies on cervical cancer, only the exons 5 to 8 were analyzed. Approximately 90% of mutations described are on this region. Recent studies on several cancer suggests that mutation frequency in the other exons must be considered. The aim of this work was to verify whether mutations on coding and non-coding regions occur in cancer tissue from cervical cancer in patients from Rio Grande do Norte using Denaturing Gradient Gel Electrophoresis (DGGE) as screening tool. Exons 8 to 11 were analyzed including some introns from 80 tumor samples and 8 peripheral blood samples from healthy women. DNA were submitted to PCR using primers with GC clamp on the end of one of them. The results were observed for each region after DGGE and silver staining. It was observed no amplified fragment with different migration profile from those obtained from DNA of peripheral blood. These results agree with those from literature where TP53 mutations in cervical cancer have been described in a very low frequencyMaster Thesis Desenvolvimento de sistemas farmacêuticos emulsionados para veiculação gênica(Universidade Federal do Rio Grande do Norte, 2007-02-26) Veríssimo, Lourena Mafra; Egito, Eryvaldo Sócrates Tabosa do; Lima, Lucymara Fassarela Agnez; ; http://lattes.cnpq.br/1083882171718362; ; http://lattes.cnpq.br/6907806915889763; ; http://lattes.cnpq.br/9173400981540256; Magalhães, Nereide Stella Santos; ; Medeiros, Sílvia Regina Batistuzzo de; ; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8Broadly speaking, the concept of gene therapy involves the transfer of a genetic material into a cell, tissue, or organ in order to cure a disease or at least improve the clinical status of a patient. Making it simple, gene therapy consists in the insertion of functional genes into cells containing defective genes by substituting, complementing or inhibiting them. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, it is a key issue to create systems able to transfer and protect the DNA until it reaches the target, the vectors. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easily produced, present controllable stability and enable transfection. The aim of this work was to develop an emulsion for gene therapy and evaluate its ability to compact nucleic acids by the development of a complex with the plasmid pIRES2-EGFP. The first step was to determine the Hydrophilic Lipophilic Balance (HLB) of the Captex® 355 (oily internal phase of the emulsion) through long and short term stability assays. Based on the results, emulsions composed of Captex® 355, Tween 20® and Span 60® with 10.7 HLB were produced by three different methods: phase inversion, spontaneous emulsification and sonication. The results showed that the lowest diameter and best stability of the emulsions were achieved by the sonication method. The cationic emulsions were made by adding DOTAP to the basic emulsion. Its association with pIRES2-EGFP was evaluated by electrophoresis. Several rates of emulsion and DNA were evaluated and the results showed that 100% of the complex was formed when the rate DOTAP/DNA(nmol/µg) was 130. In conclusion, the overall results show the ability of the proposed emulsion to compact pIRES2-EGFP, which is a requirement to a successful transfection. Therefore, such formulation may be considered a promising candidate for gene therapyMaster Thesis Levantamento cariotípico em espécies de peixes marinhos costeiros de fundo arenoso (Osteichthypes, Perciformes)(Universidade Federal do Rio Grande do Norte, 2007-02-27) Accioly, Ingrid Vilar; Molina, Wagner Franco; ; http://lattes.cnpq.br/8437464961129518; ; http://lattes.cnpq.br/9890715324172958; Affonso, Paulo Roberto Antunes de Mello; ; http://lattes.cnpq.br/4149576837587664; Blaha, Carlos Alfredo Galindo; ; http://lattes.cnpq.br/2307806644081146Cytogenetics analyses in fish are important because they compose a private group among the vertebrates, occupying a central position in the animal evolution. The Perciforms Order, dominant in the marine and freshwater environment, it constitutes a model potentially useful in the genetic evaluation of populations, as well as in the understanding of its evolutionary processes. In spite of this, cytogenetics studies in this great group is scarce, above all for the inhabitants of sandy bottom and pelagics habits. The present work proposed to contribute for the cytogenetic characterization of nine species of fish marine of sandy bottom of the coast of Rio Grande do Norte (Brazil), identifying the evolutionary patterns related to the karyotype in these species and the existence of filogenetics affinities between them and other Perciformes. The animals were collected in the beaches of the Redinha, Ponta Negra and Búzios (Coast of Rio Grande do Norte) and in Saint Peter and Saint Paul Archipelago. Later on they were submitted to the cytogenetics technical that consist of mitotic estimulation, obtaining of mitotics chromosomes, proceeded by techniques of conventional coloration (Giemsa) and chromosomic bands (Ag-RONs and C band). Diploid number and fundamental number equal to 48 were observed in most of the species: Menticirrhus americanus, Ophioscion punctatissimus, Pareques acuminatus (Sciaenidae); Chloroscombrus chrysurus (Carangidae); Echeneis sp. 2 (Echeneidae); Archosargus probatocephalus (Sparidae) and Orthopristis ruber (Haemulidae). Trachinotus goodei (NF=52) (Carangidae) and Echeneis sp. 1 (Echeneidae) (NF=54) presented variation in NF, staying constant a diploid number equal to 48. RONs was situated in pericentromeric position in whole the scianids, and in the species Echeneis sp. 2 (22° pair), O. ruber and A. probatocephalus (1° pair), coinciding with great heterocromatics blocks in M. americanus (1° pair), P. acuminatus (2° pairl) and O. ruber (1° pair). RONs was also located in the telomeric area of the short arm of the 5° and 11° acrocentrics pairs in T. goodei, 4° and 19° pairs of C. chrysurus, 1° pair (sm) of Echeneis sp. 1. The C band detected centromeric blocks in most of the chromosomes of the species of Sciaenidae, Carangidae and Echeneidae, with great blocks in A. probatocephalus (4° pair). Heterocromatic blocks in telomeric areas in submetacentrics of Echeneis sp. 1, and pericentromerics in M. americanus (1° and 8° pairs), O. punctatissimus (1° pair) and P. acuminatus (2° pair) were also observed. It is noticed a marked conservatism cromossomic in the species of the family Scianidae and Haemulidae in what says respect to the number of acrocentrics chromosomes and the location of RONs. Even so it is outstanding the presence of heterocromatinization events during the karyotypic evolution of this family. Already in the families Sparidae and Carangidae, the obtained results reaffirm examples of small variations structural resultants of inversion and translocation Robertsonian, as important mechanisms of diversification karyotipical, as well as a pattern numerical evolutionary conserved, also observed in representatives of Echeneidae of Atlantic in relation to Pacific. The presence of RONs multiple, observed in the species T. goodei and C. chrysurus seems to represent a character derived in the family Carangidae. The results for the species O. ruber and A. probatocephalus suggest the presence of possible geographical or climatic barriers among populations of NE of Brazil in relationship the one of the SEMaster Thesis Estudos in silico da proteína AlkB e prospecção de bactérias alcanotróficas em solos da bacia petrolífera potiguar(Universidade Federal do Rio Grande do Norte, 2007-02-28) Pereira, Matheus Silva; Blaha, Carlos Alfredo Galindo; ; http://lattes.cnpq.br/2307806644081146; ; http://lattes.cnpq.br/4843315104775965; Araújo, Magnólia Fernandes Florêncio de; ; http://lattes.cnpq.br/4511989641459455; Oliveira, Valéria Maia de; ; http://lattes.cnpq.br/3886687872358496Some microorganisms from virgin ecosystems are able to use petroleum it as source of carbon and energy. The knowledge of microbial biodiversity can help to reveal new metabolic systems for utilization alkanes with biotechnological importance. The aim of this study is: i) Accomplish an in silico study of the AlkB protein aimed to understand the probable mechanism involved on selectivity of alkanes in Gram positive and Gram negative bactéria. ii) prospect and analyze the response of the microbial alkanotrophics communities in soil and mangrove sediments of BPP RN and soil of Atlantic forest in the Horto Dois Irmãos Reserve area/PE using the molecular biomarker, gene alkB; with the PCR and PCR-DGGE approachMaster Thesis Avaliação da genotoxicidade das águas do lençol freático sob a Universidade Federal do Rio Grande do Norte(Universidade Federal do Rio Grande do Norte, 2007-02-28) Oliveira, Givanilson Brito de; Lima, Lucymara Fassarela Agnez; ; http://lattes.cnpq.br/1083882171718362; ; http://lattes.cnpq.br/4438490047829028; Medeiros, Sílvia Regina Batistuzzo de; ; http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8; Lemos, Clarice Torres de; ; http://lattes.cnpq.br/5348706405357207The groundwater represents the most important freshwater supply of planet. Dailly, in all world a great amount of toxic and genotoxic material reaches the aquatic systems, mainly the aquifers. The Barreiras aquifer through of five water wells is responsible for the supplying of Universidade Federal do Rio Grande do Norte (UFRN). All water wells are polluted with nitrate and some heavy metals, two of them were disabled. The genotoxicity of groundwater samples from Barreiras Aquifer in UFRN was assessed using the Allium cepa test, the Ames test and the Salmonella typhymurium microsuspension test (Kado test). For the Allium cepa test the influence of the groundwater samples collected on macroscopic (root length, colour and form) and microscopic (root tip mitotic index, chromosome aberrations and micronucleus) parameters was examined. All water samples caused a significant increase of the chromosome and mitotic aberration frequency and reduction on the rooth growth compared to negative control. Bridges and chromosome stickness were the most frequent kind of aberration in dividing cells. Furthermore, breaks were also observed. No significant increase in the number of micronuclei was found in relation to the negative controls. For Ames test were used the Salmonella typhymurium strains TA98 and TA100 without metabolic activation, applying the direct method. Prior to the Kado test, organic fractions from the water samples were obtained through XAD resin concentration. The mutagenicity organic extracts were evaluated by Kado test using TA98 and TA100 strains, in the absence and presence of S9 mix (metabolic activation). The concentrations of seven heavy metal ions were measured in water samples, but only Ni, Cu and Cr levels exceeded the permissible maximum concentration for the natural reservoirs. The results obtained for mutagenic activity using the Ames test were negative in all raw water samples analyzed. Positive results in XAD4 extracts of water samples were obtained for TA98 in the presence of S9 mix for two stations. Concentrations of heavy metals and nitrate can be correlated with the toxicity and genotoxicity of water analyzed. The mutagenic effect detected with TA98 strain suggested that organic compounds (after metabolization) are involved with the mutagenicity detected in the samples analyzed. The data set obtained in this work indicated the presence of at least two classes of mutagens: organic and inorganic compounds