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Orientador: Clarice Weis Arns / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T07:43:09Z (GMT). No. of bitstreams: 1 Silva_LucianaHelenaAntoniassida_M.pdf: 1949067 bytes, checksum: 9e01c84eac5d3921b2a96a2cb7cceb31 (MD5) Previous issue date: 2007 / Resumo: Os membros da família Paramyxoviridae, subfamília Pneumovirinae são vírus envelopados, não-segmentados dotados de genoma de RNA de fita simples com sentido negativo. O vírus Respiratório sincicial humano (hRSV) é o agente viral melhor caracterizado neste grupo, associado à doença do trato respiratório inferior. Recentemente foi identificado um novo patógeno humano pertencente à subfamília Pneumovirinae, o metapneumovírus humano (hMPV), o qual possui similaridades com o hRSV, na sua organização genômica, estrutura viral, antigenicidade e sintomas clínicos. Na primeira parte do presente trabalho o objetivo foi analisar a variabilidade genética dos isolados de hRSV circulantes na região de Campinas, no período de Abril a Setembro de 2004, comparando as seqüências previamente obtidas do gene que codifica as proteínas F e G do vírus com seqüências parciais e completas de gene homólogo de isolados identificados em outros países. A análise filogenética permitiu classificar os isolados de hRSV como pertencentes aos subgrupos A e B. Os isolados pertencentes ao subgrupo A se distribuíram em três genótipos: GA2, GA5 e SSA1. O genótipo SSA1 foi identificado pela primeira vez no sul da África, e existem poucos relatos do mesmo na literatura. As seqüências analisadas dos isolados do subgrupo B foram identificadas em 3 genótipos distintos dentro desse subgrupo, notadamente GB3 (SAB3) e BA (BAIII). Das amostras analisadas no presente estudo, duas foram identificadas como pertencentes ao genótipo BA, os isolados apresentaram a duplicação dos 60nt na posição 781-840 do gene. Na segunda parte da tese o objetivo foi identificar a presença e caracterizar molecularmente o hMPV por seqüenciamento parcial do gene que codifica para a proteína F de amostras coletadas de crianças em dois hospitais universitários na região de Campinas, São Paulo, Brasil. Com base nas seqüências de nucleotídeos do gene F do hMPV, foi identificada a presença do subgrupo B/genótipo B1 em nosso trabalho, semelhante ao que foi relatado em 2004 na Austrália. Nossas amostras apresentaram duas amostras variantes neste mesmo genótipo, com base nas seqüências de nucleotídeos / Abstract: The members of the Paramyxoviridae are enveloped, non-segmented viruses, with negative-sense single stranded genomes. Respiratory syncytial virus (hRSV) is the best characterized agent viral of this group, associated with respiratory diseases in lower respiratory tract. Recently, a new human pathogen belonging to the subfamily Pneumovirinae was identified, the human metapneumovirus (hMPV), which is structurally similar to the hRSV, in genomic organization, viral structure, antigenicity and clinical symptoms. In the first part of the present work the objective is to analyze the genetic variability hRSV isolates circulating in the region of Campinas, in the period April and September 2004, comparing previously obtained sequences from the F and G protein gene of such strains with other partial and complete gene sequences from the homologous genes from isolates identified in other countries. The phylogenetic analysis conducted here allowed us to allocate the isolates belonging to groups A and B. The isolates belonging to the group A showed three clusters, GA2, GA5 and SAA1. The genotype SSA1 was previously identified in South Africa, and there are few reports of such genotype it in the literature. Analyzed sequences belonging to group B were identified in three distinct clusters, GB3 (SAB3) and BA (BAIII). Of the analyzed sequences in the present study, two were identified as belonging to genotype BA the isolates showed 60 nucleotide (nt) duplication at positions 781-840 the gene. In the second part of the thesis the objective was to identify the presence and molecular characterization of hMPV by partially sequencing of the F protein gene in samples collected from children of two university hospitals in the region of Campinas, São Paulo state, Brazil. Based on nucleotide sequences of hMPV F gene, we identified the presence of subgroup B/genotype B1 in our work, similarly to reports in 2004 in Australia. Our samples showed to variants in the same genotype, based on nucleotides sequences / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

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Bones have a large intrinsic capacity for repair and regeneration following an injury, however, an estimated 5-10% of nearly 8 million fractures that occur every year in the United States lead to nonunions. The process of bone regeneration is a complex trait that brings together different complements of molecular and cellular interactions to carry out its necessary mechanical functions. These interactions may be attributable to the effects of genetic variations that contribute to differences in bone morphology and fracture healing. This study was undertaken to determine how genetic variability that controls phenotypic qualities of bone affect rates and patterns of fracture healing. Three inbred strains of mice (A/J (AJ), C57BL/6J (B6), and C3H/HeJ (C3)) with known structural and biomechanical differences resulting from fetal bone development were examined. Transverse fractures were generated in the femur and healing traits were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), micro-computed tomography (μCT), biomechanical torsional testing, and cartilage contrast-enhanced micro-computed tomography (CECT). The temporal analysis of gene expression revealed that B6 had the longest duration of chondrocyte maturation and the greatest relative expression of osteogenic genes relative to either C3 or AJ. While AJ and C3 exhibited similar patterns of chondrogenesis, AJ initiated osteogenesis earlier than C3. These results suggest that compared to either AJ or B6, the C3 strain exhibited the least temporal coordination between the chondrogenic and osteogenic stages. Consistent with the relative patterns of RNA expression, μCT evaluations at day 21 post fracture showed that B6 had higher callus mineralization than AJ and C3. μCT, cartilage CECT, and biomechanical testing revealed less tissue mineralization and more cartilage near the fracture gap, which indicated a less developed bony bridge in C3 calluses at day 21 post fracture. The lack of large amounts of cartilage in calluses of all strains by day 21 indicated that all strains had initiated osteogenesis by this time. Taken together, these results showed that mice with different genetic backgrounds express different patterns of mobilization and renewal of skeletal stem cells with differing temporal progressions of chondrogenic and osteogenic differentiation. These data further show that these variations affect the phenotypic properties of fracture calluses during the process of fracture healing.

Resumo: Os búfalos possuem importância econômica como animais de produção no cenário agropecuário brasileiro e mundial. O conhecimento de polimorfismos em marcadores microssatélites mapeados em búfalos é imprescindível para auxiliar em questões evolutivas da espécie, variabilidade genética intra e inter-populacional, além de serem potencialmente úteis para os programas de melhoramento animal. Foram utilizadas três raças comerciais bubalinas, Mediterrâneo, Jafarabadi e Murrah de rebanhos brasileiros para análise de polimorfismos visando avaliar a diversidade genética existente dentro e entre as raças. Foram analisados 30 animais de cada raça pela amplificação do DNA genômico extraído do bulbo de pelos utilizando quatro locos microssatélites localizados em um cromossomo de interesse econômico. Os produtos de PCR foram separados por eletroforese vertical em gel de poliacrilamida desnaturante. O loco IDVGA-53 não obteve sucesso na amplificação e foi descartado das análises de polimorfismo. Os locos MB099 e BL41 foram monomórficos. Os parâmetros de diversidade foram gerados para o loco CSSM054, o único polimórfico, com média de 3,33 alelos por loco. A heterozigosidade observada com média de 0,503 foi maior que a esperada, indicando excesso de heterozigotos. As raças Mediterrâneo e Jafarabadi apresentaram desvio de equilíbrio de Hardy-Weinberg. O parâmetro Theta indicou evidências de que as três raças diferem entre si, e quando analisadas duas a duas foi possível verificar alta estruturação populacional entre elas. A maior distância genética foi verificada entre as raças Mediterrâneo e Murrah. O conteúdo de informação polimórfica revelou que o loco CSSM054 foi altamente informativo para a raça Mediterrâneo, portanto, considerando todos os resultados, essa raça apresentou maior variabilidade genética / Abstract: The buffalo water are economically important livestock in Brazilian global and agricultural scenario. The knowledge of polymorphisms in microsatellites markers mapped in buffaloes is essential to assist in evolutionary studies of species, genetic variability within and between populations, as well as being potentially useful for animal breeding programs. Three commercial breeds of buffalos (Mediterranean, Jaffarabadi and Murrah) of Brazilian herds were used for polymorphisms analysis to evaluate the genetic diversity within and among them. In each breed 30 animals were analysed by amplification of genomic DND extracted from the hair bulbs using four microsatellite loci located on a chromosome of economic interest. PCR products were separated by vertical electrophoresis in denaturing polyacrylamide gel. The IDVGA-53 locus was not successful in amplifying and was discarded from the analysis of polymorphism. The loci MB099 and CSSM054 were monomorphic. The parameters of diversity were generated for the locus CSSM054, the only polymorphic, with an average of 3.33 alleles per locus. With a mean value of 0.503, the observed heterozygosity was higher than the expected, indicating excess of heterozygotes. Mediterranean and Jaffarabadi breeds had deviation of Hardy-Weinberg equilibrium. The parameter Theta indicated evidence that the three breeds differ and when they were analyzed in pairs, it was observed high population differentiation among them. The largest genetic distance was found between the Mediterranean and Murrah breeds. The polymorphic information content revealed that the locus CSSM054 was highly informative to the Mediterranean breed, therefore considering all the results that race had a high genetic variability / Orientador: Vera Fernanda Martins Hossepian de Lima / Coorientador: Maria Elisabete Jorge Amaral / Banca: Sandra Aidar de Queiroz / Banca: Beatriz da Costa Aguiar Alves Reis / Mestre

In this study, hypervariable region I (HVRI) of the mitochondrial DNA (mtDNA) control region, and five microsatellite loci were used to assess genetic variability and the extent of hybridization between the two fur seals (Arctocephalus tropicalis and A. gazella), that occur on Sub-Antarctic Marion Island. Both species were harvested during the 18th and 19th centuries, leading to a reduction in population size and the extinction of A. gazella at some localities. Whilst both species have recovered and are increasing in size, it is not clear to what extent sealing has affected genetic variation, although a more pronounced effect would be expected for A. gazella, given the more intensive harvesting of this species. The current study confirmed this hypothesis and revealed that A. gazella had a nucleotide diversity of 2.9 % whilst for A. tropicalis it was 4.2 %, across the HRVI mtDNA region sequenced. For microsatellite DNA, genetic variation in A. tropicalis was higher than in A. gazella in terms of the total number of alleles detected and the level of heterozygosity (HE=0.875, HO=0.845, mean number of alleles=13.6 and HE=0.799, HO=0.781, mean number of alleles=13, respectively). Diversity in both species is among the highest recorded in pinnipeds to date, and suggests that sealing did not overly affect the levels of genetic variation in these species. In terms of population structure, A. tropicalis show a high level of population structure, as indicated by the ΦST of 0.32 between Marion and Gough Island. Furthermore, the A. tropicalis haplotype tree comprising individuals from Marion, Iles Crozet, Gough, and Amsterdam islands, recovered three divergent evolutionary lineages with bootstrap values of 86% and 98%, for two of these lineages, indicating strong genetic structure and independent evolution. Shared haplotypes between Marion and other islands confirmed genetic exchanges, whilst the grouping of Marion and Gough Islands together is indicative of regular migration between these two islands. For A. gazella, the haplotype tree recovered numerous instances of grouping of individuals from Marion and Bouvetøya Islands confirming the hypothesis Bouvetøya is likely source of immigrants to Marion Island. This was further confirmed by low population differentiation between these two islands (FST = 0.062 and ΦST of 0.08). The level of hybridization between these species was low at Marion Island with only one hybrid being detected among the 134 animals for which mtDNA data were generated, corresponding to 0.75%. The same individual was identified as a hybrid, following microsatellite profiling of 146 animals, corresponding to a hybridization estimate of 0.68 %. This hybrid individual was classified phenotypically as A. gazella and genotypically was shown to have A. tropicalis ancestry. This level of hybridization is low compared to the other islands where the two species co-occur. However as the samples used in this study were primarily collected from species-specific sites, this may be an underestimate, and the studies focusing on sites where they are known to occur symaptrically, may yield higher estimates. / Dissertation (MSc)--University of Pretoria, 2009. / Zoology and Entomology / unrestricted

Submitted by (ana.araujo@ufrpe.br) on 2017-02-20T17:20:01Z No. of bitstreams: 1 Marcelo de Ataide Silva Filho.pdf: 290490 bytes, checksum: c973572d5c431d1b89267bcfca5d5b34 (MD5) / Made available in DSpace on 2017-02-20T17:20:01Z (GMT). No. of bitstreams: 1 Marcelo de Ataide Silva Filho.pdf: 290490 bytes, checksum: c973572d5c431d1b89267bcfca5d5b34 (MD5) Previous issue date: 2006-02-20 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The international market of the floriculture is presented in constant growth. In Brazil the branch of the floriculture grows to each year. One of the impediments of this expansion is the commercialization of dumb with illnesses. The embryo culture makes possible the production of free vegetal parts of illnesses, with plantations more uniforms and bigger precocity. The objective of this work was to analyze the genetic variability of changes of Heliconia bihai proceeding from culture embryos (CE) compared with the changes of vegetative propagation (PV), using the molecular marker of type RAPD. The DNA was extracted of 19 different individuals, having been ten proceeding ones from vegetative propagation and nine of embryo culture. These had been analyzed and amplified with six oligonucleotídeos of RAPD. The oligonucleotídeos of RAPD, used had demonstrated efficient in the study of the variability in H. bihai, therefore they had amplified with effectiveness and they had detected polymorphism sufficiently. The dendrograma showed that it had the formation of two groups, being in the one of this separation of two sub-groups for the type of propagation. The groupings had revealed sufficiently next when the coefficients of genetic similarity are considered. The data generated by means of RAPD had vegetative demonstrated one high similarity of the materials propagated for culture of embryo with the propagated individuals (81%), not making impracticable the propagation for culture of embryos in vitro. Our results suggest that for study of genetic variability in H. bihai, data generated through technique RAPD had been sufficiently satisfactory. The coefficient of this genetic variation is essential for the development of strategies of propagation in commercial scale of the studied vegetal material. / O mercado internacional da floricultura apresenta-se em constante crescimento. No Brasil o ramo da floricultura cresce a cada ano. Um dos entraves desta expansão é a comercialização de mudas com patógenos. O cultivo de embrião possibilita a produção de propágulos vegetais livres de doenças, com plantios mais uniformes e com maior precocidade. O objetivo deste trabalho foi analisar a variabilidade genética de mudas de Heliconia bihai provenientes de cultivo embriões (CE) comparadas às mudas de propagação vegetativa (PV), utilizando o marcador molecular do tipo RAPD. O DNA foi extraído de 19 diferentes indivíduos, sendo dez provenientes de propagação vegetativa e nove de cultivo de embrião. Estes foram analisados e amplificados com seis oligoinucleotíodeos de RAPD. Os oligonucleotídeos de RAPD utilizados demonstraram-se eficientes no estuda da variabilidade em H. bihai pois amplificaram com eficácia e detectaram bastante polimorfismo. O dendrograma mostrou que houve a formação de dois grupos, sendo em um destes a separação de dois subgrupos pelo tipo de propagação. Os agrupamentos mostraram-se bastante próximos quando considerados os coeficientes de similaridade genética. Os dados gerados por meio de RAPD demonstraram uma alta similaridade dos materiais propagados por cultivo de embrião com os indivíduos propagados vegetativamente (81%), não inviabilizando a propagação por cultivo de embriões in vitro. Nossos resultados sugerem que para estudo de variabilidade genética em H. bihai. dados gerados através da técnica RAPD foram bastante satisfatórios. O coeficiente desta variação genética é essencial para o desenvolvimento de estratégias de propagação em escala comercial do material vegetal estudado.