Conservação de material genético de espécies silvestres do bioma caatinga utilizando a manipulação oócitos inclusos em folículos ovarianos pré antrais (MOIFOPA)

The objective of the present thesis was to use the manipulation of oocytes enclosed in preantral follicles (MOEPF) as a tool for the female gametes rescue and optimization, from wild species of Caatinga biome. The thesis was divided into 4 experiments. At first experiment, it was performed the estimative and description of the agouti (Dasyprocta leporina) preantral follicles (PF) histologic and ultrastructural features, in which it was estimated 4419.8 ± 532.26 and 5397.52 ± 574.91 follicles for the right and left ovary, respectively, and the majority (86,63%) belonged to the primordial follicles category (P<0.05). Most of the population consists of morphologically normal follicles (70.78%), presenting a large and central nuclei and uniform cytoplasm. At ultrastructural evaluation it was verified the presence of a great number of round mitochondrias associated to lipid droplets. In the second experiment, it was performed the estimative and description of yellow-toothed cavies (Galea spixii) PF characteristics, also, the evaluation of the effect of solid surface vitrification (SSV) on the in situ PF morphology. The total of 416.0 ± 342.8 PF was estimated for the ovary pair and the presence of a large quantity of primary follicles (P<0.05) was evidenced. Most of the PF was morphologically normal (94.6%), in which the oocyte nuclei presented condensed granules of heterochromatin. Round or elongated shaped mitochondria constituted the most abundant organelles. In regard of the SSV, the protocol using the dimethylsulfoxide (DMSO) 3M possibility the preservation of 69.5% of morphologically normal PF, which was evidenced by the light and transmission electronic microscopy. At third experiment, the evaluation of the SSV procedure on the morphology and viability in situ PF form collared peccaries (Pecari tajacu) was performed. No differences were observed among treatments, in which the use of DMSO, ethylene glycol (EG) and dimethylformamide (DMF) as cryoprotectants, regardless its concentration, promoted the morphology preservation of much than 70% of PF. Concerning the PF viability, the DMSO and EG promoted the best preservation. The fourth experiment aimed to evaluate the effect of α MEM+ or TCM199 associated or not to 50 ng of FSHr on the morphology, activation and growth of collared peccaries PF, in vitro cultured (IVC) during 1 or 7 days and the effect on the extracellular matrix (ECM). After 7 days of IVC only the use of TCM199/FSH maintained the proportion of intact PF, similar to day 1(63.2%), however, no differences were observed among treatments (P>0.05). Also, an improvement of the proportion of intact growing PF was verified (P>0.05). By the Ag-NOR analysis it was observed that only the treatment using TCM199/FSH promoted the maintenance of cell proliferation similar to day 1 (P>0.05). The picrosirius red stain revealed that ECM remained intact in all treatments (P>0.05). Thus, as the general conclusion, the use of MOEPF in the refereed species allowed the knowledge of aspects related to its reproductive morphology and physiology, enabling the germplasm conservation, with the possibility of germplasm bank formation, as the elucidation of mechanisms related to the PF survive and in vitro development.

Metagenômica comparativa de solo de regiões de mata atlântica e caatinga do estado do Rio Grande do Norte - Brasil

The microorganisms play very important roles in maintaining ecosystems, which explains the enormous interest in understanding the relationship between these organisms as well as between them and the environment. It is estimated that the total number of prokaryotic cells on Earth is between 4 and 6 x 1030, constituting an enormous biological and genetic pool to be explored. Although currently only 1% of all this wealth can be cultivated by standard laboratory techniques, metagenomic tools allow access to the genomic potential of environmental samples in a independent culture manner, and in combination with third generation sequencing technologies, the samples coverage become even greater. Soils, in particular, are the major reservoirs of this diversity, and many important environments around us, as the Brazilian biomes Caatinga and Atlantic Forest, are poorly studied. Thus, the genetic material from environmental soil samples of Caatinga and Atlantic Forest biomes were extracted by direct techniques, pyrosequenced, and the sequences generated were analyzed by bioinformatics programs (MEGAN MG-RAST and WEBCarma). Taxonomic comparative profiles of the samples showed that the phyla Proteobacteria, Actinobacteria, Acidobacteria and Planctomycetes were the most representative. In addition, fungi of the phylum Ascomycota were identified predominantly in the soil sample from the Atlantic Forest. Metabolic profiles showed that despite the existence of environmental differences, sequences from both samples were similarly placed in the various functional subsystems, indicating no specific habitat functions. This work, a pioneer in taxonomic and metabolic comparative analysis of soil samples from Brazilian biomes, contributes to the knowledge of these complex environmental systems, so far little explored

Identificação de genes MUTM em BACs da cana-de-açucar e caracterização preliminar destes genes e seus promotores

Sugarcane has an importance in Brazil due to sugar and biofuel production. Considering this aspect, there is basic research being done in order to understand its physiology to improve production. The aim of this research is the Base Excision Repair pathway, in special the enzyme MUTM DNA-glycosylase (formamidopyrimidine) which recognizes oxidized guanine in DNA. The sugarcane scMUTM genes were analyzed using four BACs (Bacterial Artificial Chromosome) from a sugarcane genomic library from R570 cultivar. The resulted showed the presence in the region that had homology to scMUTM the presence of transposable elements. Comparing the similarity, it was observed a highest similarity to Sorghum bicolor sequence, both nucleotide and peptide sequences. Furthermore, promoter regions from MUTM genes in some grass showed different cis-regulatory elements, among which, most were related to oxidative stress, suggesting a gene regulation by oxidative stress

Análise filogenética e funcional de dois genes de reparo homólogos a AP endonuclease em cana-de-açúcar: ScARP1 e ScARP3

The genome of all organisms constantly suffers the influence of mutagenic factors from endogenous and/or exogenous origin, which may result in damage for the genome. In order to keep the genome integrity there are different DNA repair pathway to detect and correct these lesions. In relation to the plants as being sessile organisms, they are exposed to this damage frequently. The Base Excision DNA Repair (BER) is responsible to detect and repair oxidative lesions. Previous work in sugarcane identified two sequences that were homologous to Arabidopsis thaliana: ScARP1 ScARP3. These two sequences were homologous to AP endonuclease from BER pathway. Then, the aim of this work was to characterize these two sequence using different approaches: phylogenetic analysis, in silico protein organelle localization and by Nicotiana tabacum transgenic plants with overexpression cassette. The in silico data obtained showed a duplication of this sequence in sugarcane and Poaceae probably by a WGD event. Furthermore, in silico analysis showed a new localization in nuclei for ScARP1 protein. The data obtained with transgenic plants showed a change in development and morphology. Transgenic plants had slow development when compared to plants not transformed. Then, these results allowed us to understand better the potential role of this sequence in sugarcane and in plants in general. More work is important to be done in order to confirm the protein localization and protein characterization for ScARP1 and ScARP3

Desenvolvimento de nanosistemas farmacêuticos para terapia gênica

Gene therapy is one of the major challenges of the post-genomic research and it is based on the transfer of genetic material into a cell, tissue or organ in order to cure or improve the patient s clinical status. In general, gene therapy consists in the insertion of functional genes aiming substitute, complement or inhibit defective genes. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, a key issue is to create systems, vectors, able to transfer and protect the DNA until it reaches the target. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easy to produce, present suitable stability and enable transfection. The aim of this work was to evaluate two different non-viral vectors, cationic liposomes and nanoemulsions, and the possibility of their use in gene therapy. For the two systems, cationic lipids and helper lipids were used. Nanoemulsions were prepared using sonication method and were composed of Captex® 355; Tween® 80; Spam® 80; cationic lipid, Stearylamine (SA) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and water (Milli-Q®). These systems were characterized by average droplet size, Polidispersion Index (PI) and Zeta Potential. The stability of the systems; as well as the DNA compaction capacity; their cytotoxicity and the cytotoxicity of the isolated components; and their transfection capacity; were also evaluated. Liposomes were made by hydration film method and were composed of DOTAP; 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), containing or not Rhodaminephosphatidylethanolamine (PE- Rhodamine) and the conjugate Hyaluronic Acid DOPE (HA-DOPE). These systems were also characterized as nanoemulsions. Stability of the systems and the influence of time, size of plasmid and presence or absence of endotoxin in the formation of lipoplexes were also analyzed. Besides, the ophthalmic biodistribution of PE-Rhodamine containing liposomes was studied after intravitreal injection. The obtained results show that these systems are promising non-viral vector for further utilization in gene therapy and that this field seems to be very important in the clinical practice in this century. However, from the possibility to the practice, there is still a long way

Produção e avaliação de nanoemulsão catiônica como possível vetor não-viral para pIRES2-EGFP

Gene therapy is based on the transfer of exogenous genetic material into cells or tissues in order to correct, supplement or silencing a particular gene. To achieve this goal, efficient vehicles, viral or non-viral, should be developed. The aim of this work was to produce and evaluate a nanoemulsion system as a possible carrier for no-viral gene therapy able to load a plasmid model (pIRES2-EGFP). The nanoemulsion was produced by the sonication method, after been choose in a pseudo-ternary phase diagram build with 5 % of Captex 355®, 1.2 % of Tween 80®, 0.8 % of Span 80®, 0.16% of stearylamine and water (to 100 %). Measurements of droplet size, polydispersity index (PI), zeta potential, pH and conductivity, were performed to characterize the system. Results showed droplets smaller than 200 nm (PI < 0.2) and zeta potential > 30 mV. The formulation pH was near to 7.0 and conductivity was that expected to oil in water systems (70 to 90 μS/s) A scale up study, the stability of the system and the best sterilization method were also evaluated. We found that the system may be scaled up considering the time of sonication according to the volume produced, filtration was the best sterilization process and nanoemulsions were stable by 180 days at 4 ºC. Once developed, the complexation efficiency of the plasmid (pDNA) by the system was tested by agarose gel electrophoresis retardation assay.. The complexation efficiency increases when stearylamine was incorporated into aqueous phase (from 46 to 115 ng/μL); regarding a contact period (nanoemulsion / pDNA) of at least 2 hours in an ice bath, for complete lipoplex formation. The nanoemulsion showed low toxicity in MRC-5 cells at the usual transfection concentration, 81.49 % of survival was found. So, it can be concluded that a nanoemulsion in which a plasmid model was loaded was achieved. However, further studies concerning transfectation efficiency should be performed to confirm the system as non-viral gene carrier

Dificuldades de aprendizagem sobre conceitos de genética no ensino fundamental

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

Análise da genotoxicidade das águas da Lagoa da Extremoz - RN

The contamination of the waters resources for wastewater from industrial, agricultural, and domestic sources is a serious environment problem, compromising its use for human consumption and agriculture. The Extremoz-RN Lake is an important freshwater source for the supply of the city of Natal, supplying a population of approximately 160,000 habitants. This aquatic body is located near an industrial pole which can be a serious risk factor for quality of its waters. The objectives of this study were examined the genotoxicity of Extremoz Lake between September of 2006 and January of 2008, by a combination of the Allium micronucleus test, piscine micronucleus test and the comet assay in erythrocytes from peripheral blood of Oreochromis niloticus. Additionally, the level of eight different heavy metals was quantified through spectrometry of atomic absorption of flame. The Allium test did not detect increase in the frequencies of micronucleus in none of the analyzed periods, however a strong cytotoxic activity was demonstrated for decrease in mitotic index in the analyses carried in April and July of 2007. Negative results had been detected in the frequencies of micronucleus in O. niloticus. A statistic significant increase was observed in the levels of DNA damage in comet assay carried in July of 2007. The results of the chemical analysis had detected increase in the levels of cadmium, chromium, copper, nickel, lead and zinc in different periods. These results demonstrated an alteration of the water s quality of the Extremoz Lake caused for the contamination for heavy metals and increase of DNA strand breaks. The use of biomonitoring program of the heavy metal and other pollutants with genotoxic potential combinated with genotoxicity assays is recommends.

Desenvolvimento de sistemas farmacêuticos emulsionados para veiculação gênica

Broadly 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 therapy

Caracterização de uma nova exonuclease identificada em uma biblioteca metagenômica

A abordagem metagenômica tem permitido o acesso ao material genético de microrganismos não cultivados e tem sido usada para identificação de novos genes. Apesar da importância dos mecanismos de reparo de DNA para a manutenção da integridade genômica nosso conhecimento sobre mecanismos de reparo de DNA é baseado em organismos modelo como E. coli e pouco é conhecido sobre os organismos de vida livre e não cultivados. Neste trabalho, a abordagem metagenômica foi aplicada para descobrir novos genes envolvidos com a manutenção da integridade genômica. Um clone positivo foi identificado por replicar a biblioteca metagenômica em meio seletivo contendo H2O2. O clone metagenômico foi capaz de complementar parcialmente a deficiência em reparo de DNA de cepas simples e duplo-mutantes de E.coli (recA e xthA nfo, respectivamente) submetidas ao estresse gerado por H2O2 e MMS.A análise de sequência mostrou uma ORF codificando para uma proteína hipotética membro da superfamília Exo_Endo_Phos (PF03372) e, a filogenia indicou que a mesma não está inclusa em nenhuma das subfamílias EEP. Assim, uma nova nuclease foi identificada e experimentalmente caracterizada in vivo e in vitro. Ensaios específicos utilizando a nuclease purificada e oligonucleotideos fluorescentemente marcados revelaram sua atividade 3´-5´exonuclease, em substratos simples e dupla-fita, dependente de Magnésio e sensível a EDTA. Uma vez que este é o primeiro relato e caracterização de uma enzima obtida a partir de abordagem metagenômica mostrando uma atividade exonuclease, foi nomeada EXOMEG1

Metagenoma de comunidades microbianas expostas à radiação natural e a metais

Many species have specialized to live in the most varied existing environments showing the remarkable adaptability of the microbial world the most diverse physicochemical conditions. Environments exposed to natural radiation and metals are scarce around the world, presenting a microbiota still unknown. With a total number estimated between 4 and 6 x 1030 microrganisms on earth, they constitute an enormous biological and genetic pool to be explored. Metagenomic approach independent of cultivation, provides a new form to access to the potential genomic environmental samples becoming a powerful tool for the elucidation of ecological functions, metabolic profiles, as well as to identify new biomolecules. In this context, the genetic material of environmental soil and water samples from Açude Boqueirao Parelhas-RN, under the influence of natural radiation and the presence of metals, was extracted, pirosequencing and the generated sequences were analyzed by bioinformatics programs (MG-RAST and STAMP). Taxonomic comparative profiles of both samples showed high abundance of Domain Bacteria, followed by a small portion attributable to Eucaryota Domains, Archaea and Viruses. Proteobacteria, Actinobacteria and Bacterioidetes phyla showed the greater dominance in both samples. Important genera and species associated with resistance to various stressors found in region were observed. Sequences related to oxidative and heat stress, DNA replication and repair, and resistance to toxic compounds were observed, suggesting a significant relationship between the microbiota and their metabolic profile, influenced by regional environmental variables. The results of this study add valuable and unpublished data on the composition of microbial communities in these regions

Influência da radiação ionizante natural no açude do boqueirão (RN): avaliação de risco saúde/ambiente

The contamination of water bodies with toxic substances causes a decrease in water quality, representing a risk to public health. In this context, human activities are generally seen as the main sources of water degradation. However, elements found naturally in the environment can also compromise water quality. Thus, the Boqueirão‘s dam, located in the municipality of Parelhas (RN, Brazil), was chosen as area for the development of this study, as its geological region is rich in the emission of natural ionizing radiation that produces byproducts like lead and Radon. Moreover, the area has a strong human influence that enhances the risks of pollutant discharge in this body of water.Thus, the objectives of this study were centered (i) in the analysis of risk perception in the city of Parelhas (RN/Brasil) due to the use of the water from the Boqueirão Dam; and (ii) in the assessment of water quality in the Dam using methods that quantify, mainly, heavy metals and radiation levels, as well as these toxics potential of inducing mutations on genetic material. The analysis of risk perception showed that the population in the city of Parelhas can perceive a risk in using the water from the dam and that they can recognize factors that influence the water quality. Regarding the second objective, the set of data point to the contamination of the Dam by heavy metals, as well as levels of radioactive parcicles and Radon – also present in high concentrations in outdoor air and on soil. Thus, it is possible to infer that the population residing in this area is subjected to injuries caused by exposure to natural and anthropogenic contamination. Our findings corroborate with the perception of the population regarding the risks associated with the use of the Dam for several types of activities. It is expected that the information gathered in this study can substantiate activities and future researches in this semiarid region in the Rio Grande do Norte/Brazil. Also, that the set of data can enable a better understanding of the specific toxicological scenario of risk found for the population and the effect of the contamination for the biota, which aids the development of a future risk assessment and a consequent management of this local issue.

Reservatório Tabatinga (Macaíba-RN): qualidade ambiental, conflitos e uso

The reservoirs are water sources built along the fluvial basins, between rivers and dams made by concrete or earth. In Brazil they are built for different purposes, standing out the generation of energy (hydroelectric power station), flowing regulation, water reserves and flooding control, therefore they have played and still play an important role in the modern society.In the Northeastern semiarid region, they are typically used to supply cities and as a source of food.In the state of Rio Grande do Norte, the large reservoirs are intended for the same purpose.The cities settled in the riverbanks, or which have river channels crossing them, face flooding related problems. In the city of Macaíba-RN, flooding occurred systematically during the rainy season, causing great inconvenience to the local population.As product of the collective claim Tabatinga Reservoir in Jundiaí river was built, upstream of the city. Facing this background, this thesis aimed to assess the sócio-environmental quality of this reservoir.To achieve this goal, methodologies pointed to assess water quality along with the aplication of a questionnaire were used aimed to verify the quality of water and to know the perception of the residents from urban and rural área settled near to the reservoir was performed. The results showed the existence of conflicts of residents of rural communities and the presence of the reservoir, while for the city's population, the reservoir is considered not only the right solution to solve flooding in urban areas, but also as economic source for the rural population. Considering the water source assessment, this study concluded that the Tabatinga Reservoir is unfit for human use, due to the presence of metals of toxicological significance with the potential to elicit damage to the genetic material of individuals that use water from this reservoir, leading to cause serious risks to health population.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.

Caracterização de genes de reparo de DNA em cana-de-açúcar

Sugarcane is an important culture for Brazil that holds almost half of all worldwide productivity. Plants face many challenges, because of biotic and abiotic stresses presents in the production field, which could prevent plants from reaching their genetic potential. As consequence, those stresses can generate Reactive Oxygen Species – ROS – that can cause damages on DNA. Another consequence of stress is the early-flowering process, which contributes for a reduction on yield. In this context, the aim of this work is to characterize ScMUTM1 and ScMUTM2, two DNA glycosylases belonging to base excision repair pathway; and identify genes potentially related to stress and DNA repair in two sugarcane cultivars with contrasting flowering phenotypes. The characterization of the DNA glycosylases included the construction of vector to over express the recombinant proteins ScMUTM1 and ScMUTM2; they will be used in a near future to purification of these proteins and use in enzymatic assays. It was also made a phylogenetic reconstruction of this gene in plants and analysis of its promoter. With the phylogenetic analysis, it is possible to observe the presence of these genes grouped inside a branch with monocots and another one with dicots. This suggests that the duplication of this gene probably occurred after the separation of these two groups. The analysis of the promotor of MUTM shows of the presence of stress-related regulatory motifs at ScMUTM2 promoter, when compared with ScMUTM1. This may suggests that ScMUTM1 might be suffering sub functionalization process. After the analysis of microarrays data, it is observed an up-regulation from some stress-related genes in one of the conditions analyzed, related to early flowering process.