Made available in DSpace on 2014-06-11T19:32:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-07-30Bitstream added on 2014-06-13T20:23:14Z : No. of bitstreams: 1 ferreira_ia_dr_botib.pdf: 1030920 bytes, checksum: 87cbda0ccf1284061acf978c7de2a753 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A família Cichlidae tem despertado um grande interesse científico devido à rápida e extensa radiação adaptativa sofrida em alguns de seus grupos e por conter espécies com grande potencial para a aqüicultura, como a tilápia do Nilo, Oreochromis niloticus. O mapeamento físico cromossômico mostra-se promissor como ferramenta para os estudos comparativos e evolutivos entre diferentes espécies. Diante disto, o presente trabalho teve como objetivo realizar mapeamento cromossômico comparativo em ciclídeos utilizando seqüências repetitivas de DNA como sondas. Elementos transponíveis, DNA satélite e seqüências repetidas inseridas em BACs foram utilizados como sondas, através da técnica de FISH, em espécies de ciclídeos africanos e sul-americanos. Os retrotransposons Rex localizaram-se principalmente nas regiões centroméricas de espécies africanas e sul-americanas, com exceção de O. niloticus que demonstrou um padrão de localização disperso destes elementos. O acúmulo de Rex nos centrômeros destas espécies é coincidente com as regiões heterocromáticas, que representam um refúgio para seqüências repetitivas, devido à baixa taxa de recombinação. O DNA satélite SATA hibridou nos centrômeros de todas as espécies analisadas. Esta conservação centromérica mostra um papel importante destas seqüências na organização estrutural e funcional destas regiões nas diferentes espécies. Além disto, em O. karongae, foram observados sinais intersticiais em três pares cromossômicos, corroborando a hipótese de fusões cromossômicas que levaram à redução do número diplóide nesta espécie. O elemento transponível ROn-1 localizou-se intersticialmente no braço longo do par maior de O. niloticus e em posição próxima ao telômero também no braço longo do par meta-submetacêntrico (m/sm) maior dos haplocromíneos e hemicromíneos... / The Cichlidae family is one of the most species-rich families of fishes. This family has attracted the attention of the evolutionary biologists due the rapid radiation occurred in some species. Moreover, some cichlid species are important for the world aquaculture, such as Nile tilapia, Oreochromis niloticus. The chromosome mapping is useful for comparative and evolutionary studies among different species. To further understand the mechanisms of chromosome evolution in cichlids, repeated sequences were used for the comparative chromosome mapping in cichlid species. Probes containing the transposable elements (TEs) Rex1, Rex3, Rex6 and ROn-1, the SATA satellite DNA, and a BAC-clone enriched of several types of repeated DNAs were used through FISH in the chromosomes of African and South-American cichlids. The TEs Rex were mainly distributed in the centromeric region of all chromosomes in all cichlids, with the exception of O. niloticus, that presented TEs distributed overall in the chromosome arms. The localization of TEs Rex are in coincidence with heterochromatic regions, which can represent an perfect environment for the accumulation of repeated sequences. The satellite DNA was mapped in the centromeres of all cichlid species. The maintenance and centromeric distribution of the SATA satellite DNA in African cichlids suggest that this sequence can play an important role in the organization and function of the centromere in these species. Moreover, in O. karongae, the SATA have shown interstitial signals in three chromosome pairs, corroborating that chromosome fusions were involved in the reduction of diploid number in this species. The transposable element ROn-1 was localized in just one cluster in the largest chromosome of African cichlids, but in different positions, suggesting that different chromosomal rearrangements could have occurred in the origin of the largest chromosomes... (Complete abstract click electronic access below)

Made available in DSpace on 2014-10-09T12:34:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:51Z (GMT). No. of bitstreams: 0 / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Orientador: Cesar Martins / Banca: Luis Antonio Carlos Bertollo / Banca: Eliana Feldberg / Banca: Ligia Souza Lima Silveira da Mota / Banca: André Luis Laforga Vanzella / Resumo: A família Cichlidae tem despertado um grande interesse científico devido à rápida e extensa radiação adaptativa sofrida em alguns de seus grupos e por conter espécies com grande potencial para a aqüicultura, como a tilápia do Nilo, Oreochromis niloticus. O mapeamento físico cromossômico mostra-se promissor como ferramenta para os estudos comparativos e evolutivos entre diferentes espécies. Diante disto, o presente trabalho teve como objetivo realizar mapeamento cromossômico comparativo em ciclídeos utilizando seqüências repetitivas de DNA como sondas. Elementos transponíveis, DNA satélite e seqüências repetidas inseridas em BACs foram utilizados como sondas, através da técnica de FISH, em espécies de ciclídeos africanos e sul-americanos. Os retrotransposons Rex localizaram-se principalmente nas regiões centroméricas de espécies africanas e sul-americanas, com exceção de O. niloticus que demonstrou um padrão de localização disperso destes elementos. O acúmulo de Rex nos centrômeros destas espécies é coincidente com as regiões heterocromáticas, que representam um refúgio para seqüências repetitivas, devido à baixa taxa de recombinação. O DNA satélite SATA hibridou nos centrômeros de todas as espécies analisadas. Esta conservação centromérica mostra um papel importante destas seqüências na organização estrutural e funcional destas regiões nas diferentes espécies. Além disto, em O. karongae, foram observados sinais intersticiais em três pares cromossômicos, corroborando a hipótese de fusões cromossômicas que levaram à redução do número diplóide nesta espécie. O elemento transponível ROn-1 localizou-se intersticialmente no braço longo do par maior de O. niloticus e em posição próxima ao telômero também no braço longo do par meta-submetacêntrico (m/sm) maior dos haplocromíneos e hemicromíneos... (resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Cichlidae family is one of the most species-rich families of fishes. This family has attracted the attention of the evolutionary biologists due the rapid radiation occurred in some species. Moreover, some cichlid species are important for the world aquaculture, such as Nile tilapia, Oreochromis niloticus. The chromosome mapping is useful for comparative and evolutionary studies among different species. To further understand the mechanisms of chromosome evolution in cichlids, repeated sequences were used for the comparative chromosome mapping in cichlid species. Probes containing the transposable elements (TEs) Rex1, Rex3, Rex6 and ROn-1, the SATA satellite DNA, and a BAC-clone enriched of several types of repeated DNAs were used through FISH in the chromosomes of African and South-American cichlids. The TEs Rex were mainly distributed in the centromeric region of all chromosomes in all cichlids, with the exception of O. niloticus, that presented TEs distributed overall in the chromosome arms. The localization of TEs Rex are in coincidence with heterochromatic regions, which can represent an perfect environment for the accumulation of repeated sequences. The satellite DNA was mapped in the centromeres of all cichlid species. The maintenance and centromeric distribution of the SATA satellite DNA in African cichlids suggest that this sequence can play an important role in the organization and function of the centromere in these species. Moreover, in O. karongae, the SATA have shown interstitial signals in three chromosome pairs, corroborating that chromosome fusions were involved in the reduction of diploid number in this species. The transposable element ROn-1 was localized in just one cluster in the largest chromosome of African cichlids, but in different positions, suggesting that different chromosomal rearrangements could have occurred in the origin of the largest chromosomes... (Complete abstract click electronic access below) / Doutor

Made available in DSpace on 2014-10-09T12:34:13Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:01:51Z (GMT). No. of bitstreams: 0 / Este trabalho desenvolve uma metodologia para a automação da análise morfológica de imagens de cromossomos humanos irradiados no reator nuclear IEA-R1 (localizado no Instituto de Pesquisas Energéticas e Nucleares, IPEN, em São Paulo, Brasil) e, portanto, sujeitos a aberrações morfológicas. Esta metodologia se propõe a auxiliar na identificação, caracterização e classificação de cromossomos pelo profissional citogeneticista. O desenvolvimento da metodologia inclui a elaboração de um aplicativo baseado em técnicas de inteligência artificial utilizando Lógica Fuzzy e técnicas de processamento de imagens. O aplicativo desenvolvido foi denominado de CHRIMAN e é composto de módulos que contêm etapas metodológicas que suprem aspectos importantes para a obtenção de uma análise automatizada. A primeira etapa é a padronização dos procedimentos de aquisição das imagens digitais bidimensionais de metáfases através do acoplamento de uma câmera fotográfica digital comercial comum à ocular do microscópio utilizado na análise metafásica convencional. A segunda etapa é relativa ao tratamento das imagens obtidas através da aplicação de filtros digitais, armazenamento e organização das informações tanto do conteúdo da imagem em si, como das características extraídas e selecionadas, para posterior utilização nos algoritmos de reconhecimento de padrões. A terceira etapa consiste na utilização do banco de imagens digitalizadas e informações extraídas e armazenadas para a identificação dos cromossomos, sua caracterização, contagem e posterior classificação. O acerto no reconhecimento das imagens cromossômicas é de 93,9%. Esta classificação é baseada nos padrões encontrados classicamente em Buckton [1973], e possibilita o auxílio ao geneticista no procedimento de análise dos cromossomos com diminuição do tempo de análise e criando condições para a inclusão deste método num sistema mais amplo de avaliação de danos causados às células pela exposição à radiação ionizante. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Twenty-five different iso-chromosomal III lines of Drosophila melanogaster, with known haplotypes, were mixed and selected on malaoxon over twelve generations in order to find the importance of variants already present in the Drosophila genome relative to new mutations in the evolution of insecticide resistance. Measurements of mixed function oxidase and acetylcholinesterase activities, as well as insecticide resistance, were made over the twelve generations of selection and on the the iso-chromasomal III lines extracted after the selection was completed. These measurements were compared to those made of the twenty-five lines before selection. This comparison indicated that 52A, one of the origional twenty-five lines, may have been selected during this experiment. Comparisons of a possible cytochrome P-450 produced by a previously selected line of Drosophila called D23, and the DNA which is probably responsible for the production of this P-450, with the microsomal proteins and total genomic DNA of the selected lines were made using the techniques of Western and Southern blotting. The results of these procedures suggested that the mechanism of resistance used by the selected lines was not the same mechanism of resistance used by the D23 line. / Thesis / Master of Science (MSc)

International malaria control initiatives such as the Roll Back Malaria Initiative (RBM) and the Medicines for Malaria Venture (MMV) mobilize resources and spur research aimed at vector control as well as the treatment and eventual eradication of the disease. These efforts have managed to reduce incidence of malaria by an estimated 37% worldwide since 2000. However, despite the promising success of control efforts such as these, the World Health Organization reports a staggering 438,000 deaths from malaria in 2015. The continuing high death toll of malaria as well as emerging insecticide and antimalarial drug resistance suggests that while encouraging, success in reducing malaria incidence may be tenuous. Current vector control strategies are often complicated by ecological and behavioral heterogeneity of vector mosquito populations. As an additional obstruction, mosquito genomes are highly plastic as evidenced by the wealth or chromosomal inversions that have occurred in this genus. Chromosomal inversions have been correlated with differences in adaptation to aridity, insecticide resistance, and differences in resting behavior. However, a good understanding of the molecular mechanisms for inversion generation is still lacking. One possible contributor to inversion formation in Anopheles mosquitoes includes repetitive DNA such as transposable elements (TEs), tandem repeats (TRs) and inverted repeats (IRs). This dissertation provides physical maps for two important malaria vectors, An. stephensi and An. albimanus (Ch.2 and Ch. 3) and then applies those maps to the identification of inversion breakpoints in malaria mosquitoes. Repeat content of each chromosomal arm and the molecular characterization of lineage specific breakpoints is also investigated (Ch. 2 and Ch.4). Our study reveals differences in patterns of chromosomal evolution of Anopheles mosquitoes vs. Drosophila. First, mosquito chromosomes tend to shuffle as intact elements via whole arm translocations and do not under fissions or fusions as seen in fruitflies. Second, the mosquito sex chromosome is changing at a much higher rate relative to the autosomes in malaria mosquitoes than in fruit flies. Third, our molecular characterization of inversion breakpoints indicates that TEs and TRs may participate in inversion genesis in an arm specific manner. / Ph. D. / Malaria is a complex and devastating disease vectored by the bite of a female Anopheles mosquito. This disease claimed an estimated 438,000 lives in 2015. The mobilization of funding and resources as part of global malaria eradication initiatives have reduced the global incidence of malaria by 37% in the last 15 years. Deaths from malaria are also 60% lower vs. the year 2000. These promising gains are threatened by the ability of Anopheles mosquitoes to adapt in the face of malaria control efforts. Anopheles mosquito chromosomes are known to be highly plastic, as evidenced by numerous chromosomal inversions. Recent years have seen increases in insecticide resistance, and behavioral change in mosquito populations that allow them to avoid insecticides and remain prolific vectors of disease. This ability of mosquito vectors to adapt threatens to unravel recent progress towards a malaria free world. The projects presented in this dissertation explore mechanisms of chromosomal evolution, specifically the potential role of repetitive DNA in the generation of chromosomal inversions. The exploration of chromosomal inversions was facilitated by the creation of physical maps for Anopheles species. Prominent malaria vectors An. stephensi andAn. albimanus were physically mapped in Chapter 2 and Chapter 3 respectively. In chapter 1 and chapter 3 physical maps are utilized for the identification of chromosomal inversion breakpoints using 2 species (Ch. 2) and many species (Ch. 4). Repeat content was quantified along each chromosomal arm (Ch 2,4) and in inversion breakpoint regions (Ch 3). This dissertation presents physical maps for two important malaria species that have been applied to the study of chromosomal evolution and will also serve as community tools for further study of malaria mosquitoes. Our work on chromosomal evolution has revealed the Anopheles chromosomes tend to undergo translocations as intact elements and do not under fissions and fusions as seen in fruitflies. We also find that the malaria mosquito sex chromosome changes much more rapidly relative to the autosomes than in fruitflies. Additionally, repetitive DNA including transposable elements (TEs) and tandem repeats (TRs) may be encouraging chromosomal inversions but with differing roles on different chromosomal arms.

Chromosome preparations from 102 ICR albino mice were examined using a modified trypsin Giemsa staining technique. The mice were from four lines selected for maximum rate of post-weaning gain (28 generations), one line selected for minimum rate of post-weaning gain (25 generations), and two unselected control lines. Mitotic metaphase chromosome preparations were obtained from bone marrow cells of adult male and female mice. Two similar treatments were utilized in obtaining the chromosome preparations. The first treatment consisted of: 50 minute colchicine pretreatment, 30 minute hypotionic pretreatment, four-glacial acetic acid-methnanol fixation periods, 15 second trypsin period and 15 minute Giemsa staining period. The second treatment varied from the first, basically, in the length of hypotonic pretreatment, and length and number of fixation periods. A mean number of 40 telocentric chromosomes with very similar banding patterns was observed in all lines. Relative chromosome lengths for each chromosome were obtained. The lengths for the various lines, control, high and low, were compared by means of a pooled "t" test. Non-significant a₂ levels were obtained for the pairwise comparisons of the lines. Significant a₂ levels were obtained for the effects of the two treatments, trypsin and no trypsin. Karyotypes for each line were made with no chromosomal abnormalities detected in any of the lines. The selection regime followed has produced significant genetic change in several characteristics of these mice. However, these changes have apparently not been accompanied by observable alterations at the chromosomal level. / Master of Science

Malaria is the main cause of approximately one million deaths every year that mostly affect children in south of Sub-Saharan Africa. The Anopheles gambiae complex consists of seven morphologically indistinguishable sibling species. However, their behavior, ecological adaptations, vectorial capacity, and geographical distribution differ. Studying the phylogenetic relationships among the members of the complex is crucial to understanding the genomic changes that underlie evolving traits. These evolutionary changes can be related to the gain or loss of human blood choice or to other epidemiologically important traits. In order to understand the phylogenetic relationships and evolutionary history of the members of the An. gambiae complex, breakpoints of the 2Ro and 2Rp inversions in An. merus and their homologous sequence in the outgroup species were analyzed using fluorescent in situ hybridization (FISH), library screening, whole-genome mate-paired sequencing and bioinformatics analysis. Molecular phylogenies of breakpoint genes were constructed afterwards. In addition, multigene phylogenetic analyses of African malaria vectors were performed. Our findings revised the chromosomal phylogeny, and demonstrated the ancestry of 2Ro, 2R+p and 2La arrangements.  Our new chromosomal phylogeny strongly suggests that vectorial capacity evolved repeatedly in members of the An. gambiae complex, and the most important vector of malaria in the world, An. gambiae, is more closely related to ancestral species than was previously thought. Our molecular phylogeny data were in agreement with chromosomal phylogeny, indicating that the position of the genetic markers with respect to chromosomal inversion is important for interpretation of the  phylogenetic trees. Multigene phylogenetic analysis revealed that a malaria mosquito from humid savannah and degraded rainforest areas, An. nili, belongs to the basal clade and is more distantly related to other major African malaria vectors than was assumed previously. Finally, for the first time a physical map of 12 microsatellite markers for the Asian malaria vector An. stephensi was developed. Knowledge about the chromosomal position of microsatellites was shown to be important for a proper estimation of population genetic parameters. In conclusion, our study improved understanding of genetics and evolution of some of the major malaria vectors in Africa and Asia. / Ph. D.

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