Na inativação do cromossomo X (ICX) um dos dois cromossomos X presentes nas fêmeas de mamíferos placentários é silenciado transcricionalmente. Esse é um mecanismo de compensação de dose que assegura que a quantidade dos produtos gênicos oriundos do cromossomo X esteja em equilíbrio entre machos e fêmeas. A ICX pode ocorrer de modo aleatório, onde cada célula escolhe ao acaso qual será o cromossomo X inativado: cromossomo X paterno ou cromossomo X materno; ou de forma \"imprintada\" (termo adaptado do inglês imprinted), ou seja, dependente da origem parental do cromossomo X. Enquanto nas fêmeas marsupiais a inativação ocorre de forma \"imprintada\", sendo o X paterno inativado em todos os tecidos, nos mamíferos eutérios a ICX nos tecidos somáticos ocorre de modo aleatório. Porém alguns eutérios mantiveram o mecanismo \"imprintado\" de ICX exclusivamente nos tecidos extraembrionários, como ratos e camundongos. Em humanos, o estado controverso da ICX em tecidos extraembrionários foi reavaliado por nosso grupo utilizando uma análise mais ampla e identificou-se um padrão aleatório (Moreira de Mello et al., 2010), demonstrando a importância de se realizar uma análise global para se determinar o perfil de atividade do cromossomo X. Em bovinos o padrão de ICX em placenta não está claro. Ele foi verificado analisando-se a expressão de um único gene, e os autores concluíram que o padrão era \"imprintado\" (Xue et al., 2002). Porém a análise de um único gene pode não representar o estado epigenético de um cromossomo inteiro. Assim o padrão de ICX em tecidos extraembrionários bovinos se mostra uma questão importantíssima para ser esclarecida. No presente trabalho o cromossomo X bovino foi analisado em busca de SNPs (polimorfismos de base única) localizados em regiões codificadoras em genes expressos no tecido extraembrionário, permitindo assim através da análise da expressão alelo-específica determinar o padrão de expressão do cromossomo X. Os resultados apresentados neste trabalho mostram um padrão de expressão bialélica, indicando que em populações diferentes de células, diferentes cromossomos X estavam ativos. Portanto a ICX em tecidos extraembrionários bovinos ocorre de modo aleatório, padrão semelhantes àquele encontrado em humanos, e diferente daquele encontrado em ratos e camundongos. Este trabalho mostra a importância de uma análise global da expressão gênica no cromossomo X, permitindo assim traçar um perfil de atividade mais próximo possível da realidade. / In X chromosome inactivation (XCI), one of the two X chromosomes present in female mammals is transcriptionally silenced, resulting in a dosage compensation mechanism. The XCI can occur randomly, so that each cell chooses randomly which one will be the inactivated X chromosome: paternal (pX) or maternal (mX); or dependent on parental origin of X chromosome, ie, imprinted. While in female marsupials the inactivation occurs in an imprinted fashion, with the Xp inactivated in all tissues, both somatic and extra-embryonic, in the mammalian eutherians XCI in the somatic tissues occurs randomly. However some eutherians still retain the imprinted XCI mechanism exclusively in extra-embryonic tissues, such as rats and mice. In humans, the controversy of the XCI in placenta was re-evaluated by our group. Using a broader analysis, a random pattern was identified, in contrast to the previously published works. It demonstrated the importance of conducting a comprehensive analysis to determine the profile of X chromosome (Moreira de Mello et al., 2010). In cattle the pattern of XCI in bovine placenta is unclear. It was verified by analyzing the expression of a single gene, and the authors concluded that the pattern was imprinted (Xue et al., 2002). Because the analysis of a single gene may not represent the epigenetic state of an entire chromosome, the pattern of XCI in cattle extra-embryonic tissues is an important issue to be clarified. In the present study the cattle X chromosome was analyzed searching for SNPs (single nucleotide polymorphisms) located in coding regions of genes expressed in extra-embryonic tissue. So that, by analyzing the allele-specific expression it is possible to determine the X chromosome expression patter. The preset results show a bi-allelic expression pattern. This indicates that in different cells populations, different X chromosomes are active. Thus, the XCI in extra-embryonic tissues of bovines occurs randomly, similar to the human pattern but different to that verified in rats and mice. This work shows the importance of a global analysis of the gene expression in X chromosome, through which it can trace the closest activity profile as possible to reality.

Genomic imprinting is responsible for monoallelic gene expression that depends on the sex of the parent from which the alleles (one active, one silent) were inherited. X-chromosome inactivation is also a form of monoallelic gene expression. One of the two X chromosomes is transcriptionally silenced in the somatic cells of females, effectively equalising gene dosage with males who have only one X chromosome that is not complemented by a gene poor Y chromosome. X chromosome inactivation is random in eutherian mammals, but imprinted in marsupials, and in the extraembryonic membranes of some placentals. Imprinting and X inactivation have been studied in great detail in placental mammals (particularly humans and mice), and appear to occur also in marsupial mammals. However, both phenomena appear to have evolved specifically in mammals, since there is no evidence of imprinting or X inactivation in non-mammalian vertebrates, which do not show parent of origin effects and possess different sex chromosomes and dosage compensation mechanisms to mammals.¶ In order to understand how imprinting and X inactivation evolved, I have focused on the mammals most distantly related to human and mouse. I compared the sequence, location and expression of genes from major imprinted domains, and genes that regulate genomic imprinting and X-chromosome inactivation in the three extant mammalian groups and other vertebrates. Specifically, I studied the evolution of an autosomal region that is imprinted in humans and mouse, the evolution of the X-linked region thought to control X inactivation, and the evolution of the genes thought to establish and control differential expression of various imprinted loci. This thesis is presented as a collection of research papers that examines each of these topics, and a review and discussion that synthesizes my findings.¶ The first paper reports a study of the imprinted locus responsible for the human Prader-Willi and Angelman syndromes (PWS and AS). A search for kangaroo and platypus orthologues of PWS-AS genes identified only the putative AS gene UBE3A, and showed it was in a completely different genomic context to that of humans and mice. The only PWS gene found in marsupials (SNRPN) was located in tandem with its ancient paralogue SNRPB, on a different chromosome to UBE3A. Monotremes apparently have no orthologue of SNRPN. The several intronless genes of the PWS-AS domain also have no orthologues in marsupials or monotremes or non-mammal vertebrates, but all have close paralogues scattered about the genome from which they evidently retrotransposed. UBE3A in marsupials and monotremes, and SNRPN in marsupials were found to be expressed from both alleles, so are not imprinted. Thus, the PWA-AS imprinted domain was assembled from many non-imprinted components relatively recently, demonstrating that the evolution of imprinting has been an ongoing process during mammalian radiation.¶ In the second paper, I examine the evolution of the X-inactivation centre, the key regulatory region responsible for X-chromosome inactivation in humans and mice, which is imprinted in mouse extraembryonic membranes. By sequencing and aligning flanking regions across the three mammal groups and non-mammal vertebrates, I discovered that the region homologous to the X-inactivation centre, though intact in birds and frogs, was disrupted independently in marsupial and monotreme mammals. I showed that the key regulatory RNA of this locus (X-inactive specific transcript or XIST) is absent, explaining why a decade-long search for marsupial XIST was unsuccessful. Thus, XIST is eutherian-specific and is therefore not a basic requirement for X-chromosome inactivation in all mammals.¶ The broader significance of the findings reported in these two papers is explored with respect to other current work regarding the evolution and construction of imprinted loci in mammals in the form of a review. This comparison enabled me to conclude that like the PWS-AS domain and the X-inactivation centre, many domains show unexpected construction from disparate genomic elements that correlate with their acquisition of imprinting.¶ The fourth and last paper examines the evolution of CCCTC-binding Factor (CTCF) and its parologue Brother Of Regulator of Imprinted Sites (BORIS) which contribute to the establishment and interpretation of genomic imprinting at the Insulin-Like Growth Factor 2/H19 locus. In this paper I show that the duplication of CTCF giving rise to BORIS occurred much earlier than previously recognised, and demonstrate that a major change in BORIS expression (restriction to the germline) occurred in concert with the evolution of genomic imprinting. The papers that form the bulk of this thesis show that the evolution of epigenetic traits such as genomic imprinting and X-chromosome inactivation is labile and has apparently responded rapidly to different selective pressures during the independent evolution of the three mammal groups. I have introduced these papers, and discussed them generally in terms of current theories of how and why these forms of monoallelic expression have evolved in mammals.

<p>The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling.</p><p>Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors.</p><p>To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval.</p><p>The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC.</p><p>We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals.</p><p>In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.</p>

The aim of this thesis was to gain further insight into the biology of normal human skin and basal cell carcinoma (BCC). Morphology in combination with microdissection was used as primary tool for sampling. Using the X-chromosome inactivation assay, we found normal human skin to consist of a mosaic of cells, with either the maternal or the paternal X-chromosome inactivated. We believe that each tile is made up of several epidermal proliferative units with identical X-chromosome inactivation patterns. Using the same method, we found BCC to be a monoclonal neoplasm imbedded in polyclonal stroma. However, one tumor displayed clear evidence of being composed of two intermingled monoclonal tumors. To better enable molecular analysis of defined cells from tissue sections, we investigated a zinc-based fixative as alternative to neutral-buffered formalin. Zinc-based fixative preserves good quality of genomic DNA, with only slight impairment of morphology. In addition, it partly abrogates the need for antigen retrieval. The patched gene is involved in BCC development. We analyzed the distribution of a coding polymorphism (Pro/Leu) at codon 1315 in populations with different skin types. We found a reduced Pro/Pro genotype frequency in populations with lighter pigmentation. This in combination with genotype analyses of patients with multiple BCCs, showed that failure to lose the Pro allele during a shift towards lighter pigmented skin may be associated with an increased risk of developing BCC. We compared the expression profile of BCC cells with putative progenitor cells in the basal layer of epidermis. In addition to discovering several unknown genes, we found the Wnt signaling pathway to upregulated. Furthermore, differentiation markers were downregulated together with proteins important for scavenging of oxygen radicals. In conclusion, the combination of morphology, microdissection and subsequent molecular applications provided valid information deepening our understanding of normal skin and BCC.

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Copies of author's previously published works inserted. / Bibliography: leaves 321-370. / xv, 370, [21] leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of the project presented in this thesis is to isolate microsatellite markers and to construct a high resolution genetic map of the human X chromosome using these and pre-existing microsatellite markers. AC dinucleotide repeat markers are isolated from a bacteriophage library for application to the genetic localisations of X-linked disease genes, particularly those responsible for non-specific mental retardation (MRX). The genetic map is used to refine the location of the disease gene segregating in five families affected with X-linked mental retardation. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1997

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 155-182).

Thesis (Ph.D.)--Victoria University (Melbourne, Vic.), 2007.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [54]-67).

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.