Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Ecogenômica de archaea e monitoramento de comunidades de procariotos redutores de sulfato: aplicações na indústria de petróleo e gás

The human activities responsible for the ambient degradation in the modern world are diverse. The industrial activities are preponderant in the question of the impact consequences for brazilian ecosystems. Amongst the human activities, the petroliferous industry in operation in Potiguar Petroliferous Basin (PPB) displays the constant risk of ambient impacts in the integrant cities, not only for the human populations and the environment, but also it reaches the native microorganisms of Caatinga ground and in the mangrove sediment. Not hindering, the elaboration of strategies of bioremediation for impacted areas pass through the knowledge of microbiota and its relations with the environment. Moreover, in the microorganism groups associated to oil, are emphasized the sulfate-reducing prokaryotes (SRP) that, in its anaerobic metabolism, these organisms participate of the sulfate reduction, discharging H2S, causing ambient risks and causing the corrosion of surfaces, as pipelines and tanks, resulting in damages for the industry. Some ancestries of PRS integrate the Archaea domain, group of microorganisms whose sequenced genomes present predominance of extremophilic adaptations, including surrounding with oil presence. This work has two correlated objectives: i) the detection and monitoring of the gene dsrB, gift in sulfate-reducing prokaryotes, through DGGE analysis in samples of mDNA of a mangrove sediment and semiarid soil, both in the BPP; ii) to relate genomic characteristics to the ecological aspects of Archaea through in silico studies, standing out the importance to the oil and gas industry. The results of the first work suggest that the petrodegraders communities of SRP persist after the contamination with oil in mangrove sediment and in semiarid soil. Comparing the populations of both sites, it reveals that there are variations in the size and composition during one year of experiments. In the second work, functional and structural factors are the probable cause to the pressure in maintenance of the conservation of the sequences in the multiple copies of the 16S rDNA gene. Is verified also the discrepancy established between total content GC and content GC of the same gene. Such results relating ribosomal genes and the ambient factors are important for metagenomic evaluations using PCR-DGGE. The knowledge of microbiota associated to the oil can contribute for a better destination of resources by the petroliferous industry and the development of bioremediation strategies. Likewise, search to lead to the best agreement of the performance of native microbiota in biogeochemical cycles in Potiguar Petroliferous Basin ecosystem

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.

Processos carioevolutivos na ordem tetraodontiformes: uma visão através de suas diferentes linhagens

Given the great diversity of fishes, the Order Tetraodontiformes stands to show genetic and morphological characteristics enough singular. The fishes of this order have a compact DNA which favors molecular studies, as well as comparisons with more basal species. Model of genome evolution, there are still many gaps in knowledge about their chromosomal patterns and how evolutionary rearrangements influence the marked variation in DNA content of this order. In view of this, we present cytogenetic analyzes of the species Acanthostracion quadricornis (Ostraciidae), A. polygonius (Ostraciidae) Melichthys niger (Balistidae) Cantherhines macrocerus (Monacanthidae) and C. pullus (Monacanthidae), Lagocephalus laevigatus, Colomesus psittacus and Canthigaster figueiredoi (Tetraodontidae), to contribute with cytogenetic data for this group. The analysis was performed by C-banding, Ag-RONs, coloring with base-specific fluorochromes DAPI-CMA3, restriction enzymes AluI, EcoRI, TaqI, PstI and HinfI and in situ hybridization with probes for ribosomal DNA 18S and 5S. The heterochromatic ultrastructure of A. quadricornis and A. polygonius revealed a outstanding heterochromatin content, which may indicate that the accumulation or loss of extensive heterochromatin content could be responsible for large variations in genomic content displayed in different Tetraodontiformes families. The species Cantherhines macrocerus, C. pullus (Monacanthidae) and Melichthys niger (Balistidae) shows a huge karyotypic similarity both numerically and structural. L. laevigatus showed similar cytogenetic features (2n = 44 and single RONs) to the species of the genus Takifugu, which reinforces the idea of their phylogenetic relationships. C. psittacus presented the highest diploid number described for the family (2n = 56) and large amount of HC, features that related with its sister family Diodontidae. Cytogenetic analysis in C. figueiredoi revealed heterochromatic polymorphisms, RONs multiple and Bs chromosomes. These events are rare in marine fishes, and are possibly associated with the strong restructuring and genomic reduction that this family has been suffered. These features, plus the morphological and molecular data suggests that these species share the same ancestral branch, with a possible monophyletic origin. In this study, new contributions to the knowledge of evolutionary patterns facing by Tetraodontiformes are provided and discussed under cytotaxonomyc, genomic and evolutionary perspectives.

Contribuição à citogenética de testudines e análise de assimetria flutuante em Eretmochelys imbricata, Cheloniidae

This work has contributed to knowledge of the order Testudines from cytogenetic and morphological point of view. With regard to the aspects proposed cytogenetic characterization of the species Mesoclemmys tuberculata (n = 5), endemic to the Caatinga biomes, through conventional techniques of cytogenetics and molecular levels. This species presented 2n = 58, NF = 64, the first submetacentric pair, the second metacentric and third subtelocentric, and the other microchromosome telocentric. This species showed a nucleolar bearing pair, coincident with the 18S ribosomal rDNA and that proved to be heterochromatic. Small heterochromatic blocks were also found in the centromeres of the largest chromosomes, as well as terminal regions in most other chromosomes of the complement, that were GC +. Telomeric sequences showed variable patterns of signal intensity, with some repeats more intense in microchromosomes and subtly in the larger ones. When compared with other species of the genus, the G-banding patterns showed a marked similarity between them. The first karyotypic description of the species will aid in future studies and the understanding of evolutionary aspects of this family. From the morphological point of view, we carried out studies of fluctuating asymmetry in sea turtle Eretmochelys imbricata, using methods of benchmarking between hatchlings and adults and their implications for natural selection. Data were collected at two different times: first during the spawning female and the second during the outbreak and birth of the nest. The analyzed characteristics consisted of measurements of length and width of front and rear flippers (CANT, LANT, CPOS and LPOS) also collected data on the number of hull plates, side plates (NPL), the surrounding plates (NPCIRC), and plastron; plates power plants (NPP), inframarginais plates (NPIM). With the values of asymmetry we calculated the value of strict heritability for these traits, the calculation was based on only one parent. A nonparametric analysis Mann-Whitneywas performed to compare the groups (females X hatchlings, newborn hatchlings X dead hatchlings). Adult females showed no bilateral fluctuating asymmetry (FA = 0) on the number plates of the hull and plastron, while offspring, living and dead, showed a greater level of variation in these meristic parameters. In the analysis of females x hatchlings we found a significant difference between the levels of asymmetry in hoof plates (p=0.006) an the width of hindlimbs (p=0.001). Levels of FA suggest an accurate indicator as to the viability or maintenance of the individual to the reproductive phase. The coefficient of heritability (h2) of FA , obtained from the regression analysis, showed that both have low and not statistically significant values(p> 0.1). In the case of exclusion of the effective role of genetics in the generation of FA, reproductive strategies based on high number of subsidiaries products, such as those observed in E. imbricata seems to implicate the production of individuals with high level of developmental instability

Padrões cromossômicos e mapeamento de genes ribossomais 18S e 5S em peixes pelágicos Atlânticos

Cytogenetic studies in fish have been contributed significantly to a better understanding of the marine biodiversity, presenting information related to characterization, evolution and conservation of species e fisheries stocks. Among the marine species which cytogenetic data are less well known pelagic forms are detached, that despite the economic importance and conservation efforts have been suffering great pressure from the artisanal and industrial fisheries. The present work characterized cytogenetically six species of large pelagic fish in the Atlantic, belonging to the Order Perciformes, among them, four species of Scombridae, Thunnus albacares, T. obesus, Scomberomorus brasiliensis and Acanthocybium solandri and two Coryphaenidae, Coryphaena equiselis and C. hippurus using Classical cytogenetic methods as conventional staining, C-banding and Ag-NORs and molecular through staining fluorochromes AT and GC-specific and mapping of ribosomal multigene families, 18S and 5S. The identification of phylogenetic patterns and cytotaxonomic markers between the species and the presence of sex chromosomes in at least one species of Coryphaenidae, are particularly useful in the formulating of phylogenetic hypotheses, as well as comparisons between groups and populations

Evolução do reparo por excisão de nucleotídeo

The phylogeny is one of the main activities of the modern taxonomists and a way to reconstruct the history of the life through comparative analysis of these sequences stored in their genomes aimed find any justification for the origin or evolution of them. Among the sequences with a high level of conservation are the genes of repair because it is important for the conservation and maintenance of genetic stability. Hence, variations in repair genes, as the genes of the nucleotide excision repair (NER), may indicate a possible gene transfer between species. This study aimed to examine the evolutionary history of the components of the NER. For this, sequences of UVRA, UVRB, UVRC and XPB were obtained from GenBank by Blast-p, considering 10-15 as cutoff to create a database. Phylogenetic studies were done using algorithms in PAUP programs, BAYES and PHYLIP package. Phylogenetic trees were build with protein sequences and with sequences of 16S ribosomal RNA for comparative analysis by the methods of parsimony, likelihood and Bayesian. The XPB tree shows that archaeal´s XPB helicases are similar to eukaryotic helicases. According to this data, we infer that the eukaryote nucleotide excision repair system had appeared in Archaea. At UVRA, UVRB and UVRC trees was found a monophyletic group formed by three species of epsilonproteobacterias class, three species of mollicutes class and archaeabacterias of Methanobacteria and Methanococci classes. This information is supported by a tree obtained with the proteins, UVRA, UVRB and UVRC concatenated. Thus, although there are arguments in the literature defending the horizontal transfer of the system uvrABC of bacteria to archaeabacterias, the analysis made in this study suggests that occurred a vertical transfer, from archaeabacteria, of both the NER genes: uvrABC and XPs. According the parsimony, this is the best way because of the occurrence of monophyletic groups, the time of divergence of classes and number of archaeabacterias species with uvrABC system

Inferências carioevolutivas sobre grupos crípticos de peixes marinhos e estuarinos

Cytogenetic studies have been revealing a great diversity not detected, until then, in several families of fishes. Many of these groups, especially those that exhibit great diversity, like Perciformes and Siluriformes, possess species with difficult morphologic characterization, called cryptic species, commonly detected through karyotypic analyses, which reveals outstanding interespecific variations with relationship to the number and its chromosomal structures. Thus, the present work intends to contribute for the cytogenetic knowledge of marine and brackish fish species, because they peculiar life habits and by lack of cytogenetic data of your genetic aspects. Therefore, cytogenetic studies were developed in a species of Apogonidae (Perciformes), two species of sea catfishes of the family Ariidae (Siluriformes) and brackish fish Paurachenipterus galeatus (Siluriformes, Auchenipteridae), through C banding, Ag-NOR, use of base-specific flourochromes (DAPI and CMA3), as well as FISH (Fluorescent in situ hybridization) using ribosomal DNA probes 5S and 18S. The present results contribute to a better understanding of the processes of differentiation patterns and chromosome evolution in these groups. The use of other approaches (the morphology and molecular tools) will allow a larger understanding of the genetic and biological diversity of the Brazilian ichthyofauna.

Diversificação cariotípica em cinco espécies de moréias do Atlântico Ocidental (Anguilliformes)

The 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 fissions