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

Mapeamento cromossômico de DNAs repetitivos com fins biotecnológicos em peixes marinhos e interesse comercial - Rachycentridae e Lutjanidae

The Rachycentron canadum species, commonly known as beijupirá or cobia is the only representative of Rachycentridae family which has been increasingly used in marine fish farming, in intensive cultivation. As advantageous features it has easy adaptation, prolific behavior, early growth in captivity and high commercial value. Additionally, specie of Lutjanidae family (Lutjanus synagris, Lutjanus jocu, Lutjanus analis, Lutjanus alexandrei and Ocyurus chrysurus) represents an important fisheries resource in all areas of its occurrence. In Brazil, the commercial exploitation of Lutjanidae which begun in the 60's and 80's, already has showed a decline in catch volumes. This fact suggests that the snappers must have a conservative management. Despite the economic potential, little is known about the genetic and cytogenetic characteristics of these species, especially with respect to repetitive DNA analysis, which represents the major part of the eukaryotes genome, playing important evolutionary roles in the fish genome. Cytogenetic data is increasingly being used in population studies and biotechnological purposes in fishes. The cytogenetical analyzes were performed using classical methods such as Giemsa staining, C-banding and Ag-NORs, fluorochromes base-specific staining (DAPI and MM) and physical mapping of repetitive sequences among which, telomeric sequences, transposons (Tol2), retrotransposons (Rex1 and Rex3), repetitive DNA (microsatellites and Cot-1) and transcriptionally active regions of the 18S and 5S ribosomal genes and histone (H3 and H2BA) by in situ hybridization with fluorescent probes (FISH). The chromosomal patterns obtained contributed to the organization of repetitive sequences in the genome of the species, as well as karyotypical differentiation. Unusual patterns of histone sequences expansion depict the first occurrence in marine fishes. The obtained data provided subsides to the genetic knowledge of the important fisheries resource represented by the species here analyzed, seeking the marine pisciculture improvement.

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

Haemulidae, modelo cariotípico de estase evolutiva

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Parâmetros citogenéticos de espécies comerciais de carangidae (perciformes), com vistas a sua empregabilidade na conservação biológica e piscicultura marinha

Worldwide, families Carangidae and Rachycentridae represent one of the groups most important commercial fish, used for food, and great potential for marine aquaculture. However, the genetic bases that can underpin the future cultivation of these species, cytogenetic between these aspects are very weak. The chromosomal patterns have provided basic data for the exploration of biotechnological processes aimed at handling chromosomal genetic improvement, such as induction of polyploidy, androgenesis and ginogenesis, as well as obtaining monosex stocks and interspecific hybridizations. This paper presents a comprehensive cytogenetic survey in 10 species, seven of the family Carangidae and the monotypic family Rachycentridae. Classical cytogenetic analysis and in situ mapping of multigene sequences were employed, and additionally for the genus Selene and morphotypes of Caranx lugubris, comparisons were made using geometric morphometrics. In general, conservative species exhibit a marked chromosome number (2n=48). Although present in large part, different karyotypic form, retain many characteristics typical of chromosomal Order Perciformes, the high number of elements monobrachyal, Ag-NORs/18S rDNA sites and heterochromatin simply reduced, preferably centromeric. The main mechanisms involved in karyotypic diversification are the pericentric inversions, with secondary action of centric fusions. In addition to physical mapping and chromosome detail for the species are presented and discussed patterns of intra-and interspecific diversity, cytotaxonomic markers. This data set provides a better understanding of these patterns caryoevolutyonary groups and conditions for the development of protocols based on Biotechnology for chromosomal manipulation Atlantic these species

Caracterização fenotípica e genotípica de Staphylococcus aureus resistentes à meticilina isolados na cidade do Natal/RN

Staphylococcus aureus resistente à meticilina (MRSA) é um dos principais agentes de infecções associadas a serviços de saúde em todo o mundo. No Brasil, há a predominância de um clone de MRSA multirresistente denominando clone epidêmico brasileiro (CEB). Entretanto, novos clones nãomultirresistentes com alta virulência têm sido descritos em infecções comunitárias e hospitalares. O objetivo desse estudo foi realizar a caracterização fenotípica e genotípica de cepas de MRSA isoladas na cidade do Natal/RN. Inicialmente avaliamos 60 amostras de S. aureus quanto a resistência à meticilina através de diferentes técnicas fenotípicas, utilizando a detecção do gene mecA por PCR como padrão. O antibiograma de todas as cepas foi realizado utilizando 12 antimicrobianos conforme descrito pelo CLSI. As cepas de MRSA foram caracterizadas geneticamente através da tipagem do cassete cromossômico estafilocócico mec (SCCmec) e da eletroforese em campo elétrico alternado (PFGE). Dos 60 S. aureus estudados, 45 foram resistentes à meticilina. Observamos que para algumas cepas de MRSA os testes de triagem em ágar com 6μg/mL de oxacilina e difusão em meio sólido com oxacilina-1μg apresentaram dificuldades na sua interpretação. No entanto, todas as 45 amostras de MRSA, foram facilmente detectadas pelos testes com o disco de cefoxitina-30μg e pesquisa da PBP2a. A análise molecular das cepas de MRSA mostrou 8 padrões distintos de PFGE (A-H), com predominância do padrão A (73%), relacionado ao CEB. Estas carreavam o SCCmec tipo IIIA, e apresentaram uma considerável variedade de subtipos (A1-A16). Cinco cepas de MRSA portando SCCmec IV também foram xiv identificadas, três delas relacionadas geneticamente ao clone USA800 (Padrão B). Destas cinco, três (2 padrão F e 1 padrão B) foram altamente susceptíveis as drogas testadas, entretanto, dois outros isolados, padrão B, apresentaram multirresistência. As amostras restantes pertenciam a padrões de PFGE distintos dos clones internacionais predominantes em nosso continente. Para realização deste projeto de pesquisa, a metodologia exigiu a interação com pesquisadores de áreas como: infectologia, microbiologia e biologia molecular. Portanto, esta dissertação apresentou um caráter de multidisciplinaridade e transdiciplinaridade no seu desenvolvimento

Liofilização como ferramenta de preservação de sêmen humano

Liofilização é um método alternativo de preservação de espermatozóide, com vantagens sobre o criopreservado pelo baixo custo de armazenamento, facilidade no transporte pela não necessidade de containers e baixas taxas de contaminação. Esse estudo foi realizado no intuito e testar a eficácia de 5 açúcares na proteção do material cromossômico (DNA) de espermatozoides humanos liofilizados após reidratação. Para isso foram utilizados espermatozoides de indivíduos classificados como normozooespérmicos segundo OMS. Em eppendorfs foram colocados 1ml de sêmen que foram diluídos (1:1) em 1 ml de solução de Glicose, Lactose, Maltose, Manitol e Sorbitol, separadamente, nas concentrações 0.2M, 0.4M, 0.5M e 0.6M. Essas amostras foram analisadas em duas fases a primeira após congelamento observando a porcentagem de células com danos de membrana e outra análise após a liofilização, utilizando o Teste Cometa para detectar as taxas de fragmentação de DNA dos espermatozoides recuperadas após reidratação. O processo de liofilização de espermatozoides não foi capaz de recuperar a motilidade dos mesmos, mas foi capaz de manter 62.30 ± 13.76% de membrana plasmática intacta na presença de 0.6M manitol. As substâncias que melhor preservaram o DNA dessas células foram 0.2 lactose, 0.2M e 0.6M manitol e sorbitol, com apenas 4% de danos ao DNA. Esses resultados sugerem uma boa proteção ao DNA de espermatozoides humanos liofilizados, mas ainda requer testes adicionais para avaliar a fertilização e estudos que garantam a viabilidade da prole

Análise citogenética de células-tronco derivadas do subendotélio da veia umbilical humana

Mesenchymal stem cells (MSCs) are known as a population of multi-potential cells able to proliferate and differentiate into multiple mesodermal tissues including bone, cartilage, muscle, ligament, tendon, fat and stroma. Several applications of the study of EC can be emphasized the therapeutic techniques such as guided bone regeneration by implantation of EC in the affected site, without the need for bone grafts, using titanium as a vehicle. The process of cryopreservation is essential for the maintenance of cell cultures, since the cell line is frozen, it can be maintained in liquid nitrogen for an indefinite period and then thawed for therapeutic or experimental purposes. The aim of this study was to isolate a population of MSCs derived from the subendothelium of the umbilical vein human (MSCs-SUVH) to assess cytogenetic analysis by the possibility of appearance of chromosomal changes in two different situations: MSCs-SUVH regarding the process of cryopreservation and MSCs-SUVH grown on the surface of titanium. Flow cytometry analysis revealed that, this cell population was positive for the markers CD29, CD73 and CD90, but there was no expression of hematopoietic lineage markers, such as CD14, CD34 and CD45 and demonstrated capacity for osteogenic differentiation. The chromosomes obtained from the primary culture of MSCs-SUVH were analyzed by GTW banding technique, and results are described as guidelines to ISCN 2005. There was not the emergence of clonal chromosomal changes in the MSCs-SUVH in different situations analyzed. However one of the strings presented a balanced paracentric inversion, probably a cytogenetic constitutional alterations, which was present before and after the experimental situations that the MSCs-SUVH was submitted

Levantamento cariotípico em espécies de peixes marinhos costeiros de fundo arenoso (Osteichthypes, Perciformes)

Cytogenetics 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 SE

Padrões cariotípicos em tetraodontiformes (Osteichthypes) :redução genômica e mecanismos de diversificação

The Tetraodontiformes order is composed for about 400 species of fish, distributed in ten families, with circuntropical distribution. The morphologic diversity of each family reflects in, part, the different levels of specialization. This group represents an ancestry after-Perciformes and constitutes the last branch of the diffusion of the Teleósteos, occupying a position of prominence. The phylogenetics relationships of the Tetraodontiformes exist diverse works examining and, in all, these families are recognized as groups brothers, being Diodontidae next to Tetraodontidae and Balistidae next to Monacanthidae. Although it possesss a representative number of species, the works involving of the families Balistidae and Monacanthidae are few exemplary, especially species of oceanic islands. In this work cytogenetic studies in five species had been analyzed Cantherhines macrocerus, Cantherhines pullus (Monacanthidae), Melichthys niger (Balistidae), Sphoeroides testudíneus (Tetraodontidae) and Chilomycterus antennatus (Diodontidae); through conventional coloration, Ag-NORs and C banding. Ahead of the different karyological trends of evolution presented by the Tetraodontiformes, the present work also searched to verify the relation existence enters the total size of the chromosomes with the amount of DNA in these groups of Tetraodontiformes. For such, they had been correlated the total size of the chromosomes of these species, with values of content of available DNA in literature. The cytogenetics analyses for the species C.macrocerusJ C.pullus (Monacanthidae) and M.niger (Balistidae), had disclosed 40 chromosomes, all acrocentrics. All possess only one pair of NORs and pericentromeric heterochromatin. For S.testudíneus the found dyploid number was equal 2n=46, with NF=78 (16m+18sm+8st+4a), while that for C.antennatus it possesss 2n=50, with NF=76 (4m+22st+24a). Both species possess simple NORs and pericentromeric heterochromatin blocks. In M.niger, the presence of positive marking (heterochromatin and NOR) in the secondary constriction in the second chromosomic pair suggesting the occurrence of a rearrangement, possibly a fusing involving these homologous ones, indicating that these events had been important for the establishment of the karyological history of this group. A maintenance of the chromosomic constancy found in the populations of C.macrocerus (Monacanthidae) and S.testudineus (Tetraodontidae) perhaps if must for the aiding of the gene flow through oceanic chains. These data contrast with the differentiated kinds of chromosomes of C.antennatus between the Northeast coast and Southeastern, suggesting that the ecological standards of each species, added to the conditions of the marine environment, can be responsible for the karyological delineation of each species. The found characteristics for the species C.macrocerus, C.pullus, M.niger, S.testudineus and C.antennatus add it the available data for other species of Tetraodontiformes. From the data gotten in the present study, it can be inferred that the DNA content possesss direct relation with the total length of the chromosomes

Validação de espécies de Centropomus (Centropomidae, Perciformes) do litoral do Rio Grande do Norte através de caracterização citogenética e molecular

The Centropomidae family consists of three genera, Centropomus, Lates and Psammoperca. Centropomus is the most diverse group, with six Centropomus species occur in the Western Atlantic Ocean C. poeyi Chávez, 1961, C. parallelus Poey, 1860, C. mexicanus Bocourt, 1868, C. pectinatus Poey, 1860 and C. ensiferus Poey, 1860. Some of these species are considered cryptic, because of its morphological traits showed low resolution for identification purposes. Despite showing great interest as a natural resource and fish culture, aspects of their diversity and karyotypic patterns are poorly understood. In this work morphological identification and comparison of mitochondrial 16S gene sequence were used to identify the species of the genus Centropomus occurring in Rio Grande do Norte, northeastern Brazil. Two sepecies were identified, C. undecimalis and C. mexicanus, which had the chromosomal aspects analyzed, through Classical cytogenetic method analyzes (conventional staining, C-banding, Ag-NORs), fluorochrome staining AT- and GC-specific, replication bands by incorporating of the base analog 5-Bromo-2’-deoxyuridine (5-BrdU), in situ chromosomal mapping of (TTAGGG)n sequences and in situ chromosome mapping 18S and 5S rRNA genes. Both species show 2n=48 acrocentric chromosomes, with ribosomal sites (Ag-NOR/18S rDNA/ Mitramycin+) in second chromosomal pair, in telomeric position on the long arm in C. mexicanus and interstitial in C. undecimalis. The nuclear organization pair (pair 2) shown a resolutive cytotaxonomic marker for these two species. The generated data reveal a lower species diversity than previously believed, suggesting that greater attention should be paid in taxonomic identification of the species, in view of optimize commercial actions exploitation, biological conservation and cultivation.

Haemulidae, modelo cariotípico de estase evolutiva

Conselho Nacional de Desenvolvimento Científico e Tecnológico

Parâmetros citogenéticos de espécies comerciais de carangidae (perciformes), com vistas a sua empregabilidade na conservação biológica e piscicultura marinha

Worldwide, families Carangidae and Rachycentridae represent one of the groups most important commercial fish, used for food, and great potential for marine aquaculture. However, the genetic bases that can underpin the future cultivation of these species, cytogenetic between these aspects are very weak. The chromosomal patterns have provided basic data for the exploration of biotechnological processes aimed at handling chromosomal genetic improvement, such as induction of polyploidy, androgenesis and ginogenesis, as well as obtaining monosex stocks and interspecific hybridizations. This paper presents a comprehensive cytogenetic survey in 10 species, seven of the family Carangidae and the monotypic family Rachycentridae. Classical cytogenetic analysis and in situ mapping of multigene sequences were employed, and additionally for the genus Selene and morphotypes of Caranx lugubris, comparisons were made using geometric morphometrics. In general, conservative species exhibit a marked chromosome number (2n=48). Although present in large part, different karyotypic form, retain many characteristics typical of chromosomal Order Perciformes, the high number of elements monobrachyal, Ag-NORs/18S rDNA sites and heterochromatin simply reduced, preferably centromeric. The main mechanisms involved in karyotypic diversification are the pericentric inversions, with secondary action of centric fusions. In addition to physical mapping and chromosome detail for the species are presented and discussed patterns of intra-and interspecific diversity, cytotaxonomic markers. This data set provides a better understanding of these patterns caryoevolutyonary groups and conditions for the development of protocols based on Biotechnology for chromosomal manipulation Atlantic these species