Typewritten copy Includes bibliographical references

Thesis (Master of Ag. Sc.)--University of Adelaide, 1959. / Typewritten copy. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves [107]-116).

Human Artificial Chromosomes (HAC) are fascinating extrachromosomal molecules that stay independently from the host genome and are capable of segregating as efficiently as endogenous chromosomes. It has been proven that HAC are potential tools for both basic chromosome behavioural research and agents for gene therapy purposes. My DPhil project is divided into two main themes. The first theme was to develop a novel artificial chromosome in mouse embryonic stem cells. The second theme was to understand the factors affecting chromosome stability which may also affect the efficiency of artificial chromosome formation. so that our protocol for better HAC preparation can be refined. There are six results chapters in my thesis. The first three chapters described how I developed human artificial chromosomes in mouse embryonic stem cells. Initially, vectors containing a long stretch of human alphoid DNA were delivered to mouse cells using the Herpes Simplex Virus-I (HSV-l) amplicon system but the efficiency was low. Next, mouse pericentromeric and centromeric DNAs were employed for mouse artificial chromosome (MAC) via HSV-l system. However, the efficiency remained the same. Finally, I used the Microcell-Mediated Chromosome Transfer (MMCT) system to transfer HAC from HTl 080 cells into mouse ES cells and successfully established HAC in ES which were highly stable. The results obtained in this first part of my thesis suggested that to increase HAC formation efficiency it would be necessary to improve the techniques of HSV-I delivery and MMCT. Moreover, it would also be important to better characterize factors affecting chromosome behaviour. The last three results chapters focus on factors affecting chromosomes stability and improving the HSV -1 delivery system and MMCT. I undertook an in vivo study of whole cell fusion experiments with the aid of live cell irnaging system, and found that histone H2B proteins underwent a dynamic assembly/disassembly processes. Live cell imaging of MMCT suggested that the microcell delivery is a very slow process and the results may lead to a refinement of the MMCT protocol. I found it is possible to generate a single HAC using two HSV-l amplicons containing two different constructs, potentially doubling the HSV-l HAC capacity from 150 kb to 300 kb. The last chapter illustrated how the expression of non- coding centromeric satellites impaired chromosome stability in both human cultured and human embryonic stem cells. The findings revealed that non-coding centromeric RNA plays an important role on chromosome stability that might be important for artificial chromosome development.

At the core of evolutionary biology stands the study of divergence between populations and the formation of new species. This dissertation applies a diverse array of theoretical and statistical approaches to study how chromosomes evolve. In the first chapter, I build models that predict the amount of neutral genetic variation in chromosomal inversions involved in local adaptation, providing a foundation for future studies on the role of these rearrangements in population divergence. In the second chapter, I use a large dataset of the geographic variation in frequency of a chromosomal inversion to infer natural selection and non-random mating, revealing that this inversion could be implicated in strong reproductive isolation between subpopulations of a single species. In the third chapter, I use coalescent models for recombining sex chromosomes coupled with approximate Bayesian computation to estimate the recombination rate between X and Y chromosomes in European tree frogs. This novel approach allows me to infer a rate so low that would have been hard to detect with empirical methods. In the fourth chapter, I study the theoretical conditions that favor the evolution of a chromosome fusion that reduces recombination between locally adapted alleles. / text

Orientador: Louis Bernard Klaczko / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-08T11:40:37Z (GMT). No. of bitstreams: 1 Rocha_FelipeBastos_M.pdf: 1521732 bytes, checksum: 2e105d0f1d7044bc42e2f93125f6ac49 (MD5) Previous issue date: 2007 / Resumo: Drosophila mediopunctata é uma espécie pertencente ao grupo tripunctata, que tem como traço marcante um padrão de pigmentação abdominal, sob a forma de três pintas na região mediana dos últimos tergitos. Nesta espécie, este padrão é variável, havendo indivíduos com quatro fenótipos, que vão de zero a três pintas. Já se observou que esta variação tem determinação genética, com marcada influência do cromossomo II, e alta plasticidade fenotípica em resposta à temperatura de desenvolvimento. Neste trabalho, buscou-se caracterizar parte destas duas fontes de variação. Por um lado, foram estudadas as normas de reação da pigmentação a um gradiente térmico, investigando-se classes fenotípicas contrastantes. Devido ao desenho experimental, que buscou separar os efeitos desta variável de um possível papel das inversões do cromossomo II, foi possível evidenciar um forte efeito das classes fenotípicas utilizadas sobre a resposta das estirpes ao gradiente térmico, independente do cariótipo. Foram descritos, por polinômios, dois tipos de norma de reação relacionados ao fenótipo, ambos com forma de parábola, mas diferindo em relação ao coeficiente de curvatura. O grupo de estirpes de pigmentação clara apresentou uma curva côncava e o grupo escuro uma curva convexa. A norma de reação da taxa de desenvolvimento de ovo a adulto foi caracterizada a partir do mesmo procedimento. Entretanto, apesar dos efeitos significativos do cariótipo e da classe fenotípica, a homogeneidade das normas de reação descritas por regressões lineares não possibilitou uma interpretação clara destes efeitos. A plasticidade do caráter também foi investigada quanto ao período de desenvolvimento termo-sensível. Assim, foi possível determinar a porção final da fase de pupa como o período no qual ocorre a influência da temperatura sobre o fenótipo de pintas do adulto. Por outro lado, em relação à determinação genética do caráter, foram obtidas estimativas de herdabilidade para o número de pintas abdominais, em condições quase naturais. Visando estabelecer um parâmetro de comparação com outros trabalhos, foi estimada a herdabilidade do tamanho do tórax a partir do mesmo material. Os resultados deste experimento, apresentaram grande contraste entre os dois traços: as estimativas foram baixas ou não significativas para o tamanho do tórax e, em geral, altas e significativas para o número de pintas / Abstract: Drosophila mediopunctata belongs to the tripunctata species group, which has a typical abdomen pigmentation pattern, consisting of three dark spots in the last tergites. In this species, this pattern is variable, with the phenotypes ranging from zero to three spots. It has been noted that this variation has genetical determination, with strong influence from the second chromosome, and high phenotypic plasticity in response to the developmental temperature. In this work, we attempted to describe part of these two variation sources. On one side, the pigmentation reaction norm to a thermal gradient was studied, by investigating the influence of contrasting phenotypical classes. Given the experimental design, which was planned to separate the effects of this variable from a possible influence of the second chromosome inversions, it was possible to detect a strong effect of the phenotypical classes on the lineages response to the thermal gradient, independent of the kariotype. Two types of reaction norms, related to the phenotype, were detected and described by polynomial adjustment. Both had a parabolic shape, but with different curvature coefficients. The light pigmentation lineage group showed a concave curve, and the dark group had a convex curve. The reaction norm of development rate from egg to adult was described according to the same procedure. However, despite the significant effects of the karyotype and phenotypical classes, the homogeneity of reaction norms, described by linear regression, hindered a clear interpretation of these effects. The character plasticity was also investigated in respect to the developmental thermosensitive period. Thus, it was possible to determine that the period in which the temperature influence on the adult phenotype occurs is the last portion of the pupal phase. On another side, relative to the character genetic determination, heritability estimates for the number of abdominal spots were obtained, in nearly natural conditions. Aiming to establish a comparison parameter with other studies, the heritability of thorax length was estimated based on the same material. The results of this experiment reveal a great contrast between these trait estimates: for the thorax they were low or non-significant, and, in general, for the abdominal spot number, they were high and significant / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular