OBJETIVO: A perda de heterozigosidade (LOH) é capaz de avaliar as alterações genéticas de lesões potencialmente malignas. Este ensaio avalia as regiões cromossômicas polimórficas que estão próximas ou na região dos oncogenes e genes supressores de tumor conhecidos. Os objetivos desta tese foram três principais: 1) Avaliar a frequência de perda de heterozigosidade de leucoplasias bucais com diferentes graus de severidade histopatológico em regiões cromossômicas próximas aos genes supressores de tumores. 2) Comparar e correlacionar o perfil de perda de heterozigosidade entre indivíduos da British Columbia (Canadá) e Rio Grande do Sul (Brasil). 3) Avaliar os danos ao DNA que podem ocorrer durante o processamento e armazenamento das amostras de tecido parafinado. MÉTODOS: Amostras de leucoplasia bucal (com e sem displasias), fixadas em formalina tamponada 10% e parafinadas, obtidas nos laboratório de patologia bucal do Canadá e do Brasil foram selecionadas e microdissectadas. Procedeu-se a extração de DNA, amplificação por PCR das seguintes regiões microssatélites: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) e 17p13.1 (tp53 e D17S786). Após o produto do PCR foi separado e visualizado em gel de poliacrilamida por autoradiografia. RESULTADOS: Observou-se uma forte correlação entre o perfil de perda de heterozigosidade entre indivíduos com leucoplasia bucal de ambos os países, independentemente da etnicidade. Além disso, pode-se notar que amostras de tecidos parafinados submetidos a mais de 24 horas de fixação em formalina tamponada 10% não serão, em sua maioria, boas amostras para análises de DNA. CONCLUSÃO: As lesões potencialmente malignas, provavelmente não são influenciadas em sua etiopatogênia pelas diferenças étnicas. O modelo de risco genético validado por Zhang e colaboradores (2012) parece ser aplicável em nossa comunidade, sendo necessário a sua validação, respeitando procedimentos técnicos padronizados. Ainda, vale ressaltar, que é imprescindível que a comunidade científica passe a adotar metodologias que preservem o material genético das peças dos bancos de tecidos parafinados, que são de inestimável valor para a pesquisa biomédica. / OBJECTIVE: Loss of heterozygosity (LOH) can evaluate genetic alterations of pre-malignant lesions. This assay evaluates the chromosomal polymorphic regions that are present in tumor suppressor genes and oncogenes. The main objectives of this thesis were: 1) Evaluate the frequency of LOH of oral leukoplakias with different histopathological degrees at chromosomal regions of tumor suppressor genes. 2) Compare the profile of LOH between individuals from British Columbia (Canada) and Rio Grande do Sul (Brazil). 3) Evaluate the DNA damage that may occur with FFPE (formalin-fixed paraffin-embedded) tissues. METHODS: FFPE samples of oral leukoplakia (with and without dysplasia), obtained in Canadian and Brazilian oral pathology laboratories were selected and microdissected. DNA extraction and PCR amplification of the following microsatellite regions were conducted: 4q (D4S243, FABP2), 9p21 (IFNA, D9S171, D9S1748, D9S1751), 17p11.2 (CHRNB1) and 17p13.1 (tp53 and D17S786). PCR products were separated and visualized on polyacrylamide gel by autoradiography. RESULTS: A strong correlation between the LOH profile among individuals with oral leukoplakia from both countries was observed, regardless ethnicity. Furthermore, FFPE tissues subjected to more than 24 hours of fixation in 10% buffered formalin are not, generally, good samples for DNA analysis. CONCLUSION: Pre-malignant lesions etiopathogenesis may not be influenced by ethnicity. The genetic risk model validated by Zhang et al. (2012) seems to be applicable in our community, requiring its own validation, respecting standardized procedures. Still, it is important to emphasize that it is imperative that a scientific community adopts methodologies that preserve the genetic material FFPE tissues that are an invaluable resource for biomedical research.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Breast cancer (BC) is one of the most common malignancies in women. Death results from treatment failure and metastatic disease. Thousands of lives might be saved if it was possible to detect and eliminate occult metastatic cells before they become clinically evident. Therefore, there is a critical need to identify new markers to improve treatment options for these patients. Genomic instability is the earliest indication of breast cancer and the use of genomic methodologies is a progress towards early detection and treatment, through the identification of biomarkers that can be translated into novel therapy targets. The interferon regulatory factor-1(IRF-1) gene, localized on chromosome 5q31.1, is believed to act as a tumor suppressor gene in breast cancer. The IRF-1 was found to be inactivated by single nucleotide polymorphism (SNP) in breast cancer suggesting that the loss of its function might be critical to the development of the disease. The phosphatidylinositol 3-kinase (PIK3) signaling pathway mediates key cellular functions and alterations of genes in this pathway, including PIK3CA, serine-threonine protein kinases (AKT1and AKT2), phosphatase and tensin homolog (PTEN), fibroblast growth factor receptor 2 (FGFR2) and ERBB2, whose expression have been demonstrated to be altered in breast cancer patients. In addition, these genes are linked to treatment resistance. vi In this study, we have investigated allelic loss of IRF-1 gene in primary tumors obtained from patients undergoing mastectomy at Groote Schuur hospital (Cape Town, South Africa). These samples were then further analyzed for the DNA copy number changes of specific genes involved in the PIK3/AKT signaling pathway. Statistical analysis has been performed in order to correlate genomic findings with clinical-histopathological and follow up information from the patients and to establish whether these genes can predict prognosis. Our data analysis has indicated that 46 cases (45.5%) out of 101 cases were informative for the IRF-1 dinucleotide marker used for LOH analysis (Figure 3.1). LOH was detected in 23 of these informative cases (23/46; 50%). No statistical significance was found between LOH at the IRF-1 locus and age (≤50 years or >50 years) (P value = 1.0000) and earlier stage (Stages I and II) (P value= 0.4982) based on Fisher’s exact test. Patients presented a high level of DNA copy number changes in genes involved in the PIK3/AKT pathway. The most frequent changes were observed in the PIK3CA and PTEN genes. PIK3CA presented high copy number in 36.8% of the cases. PTEN was observed with low copy number in 47.5% of the cases. This dissertation shows the effectiveness of genomic methodologies as means for the detection of early breast cancer progression in South African women. The PIK 3/AKT genes can validate the usefulness of breast cancer therapies.

Epigenetic differences between maternally inherited and paternally inherited chromosomes, such as CpG methylation, render the maternal and paternal genome functionally inequivalent, a phenomenon called genomic imprinting. This functional inequivalence is exemplified with imprinted genes, whose expression is parent-of-origin specific. The dosage of imprinted gene expression is disrupted in cells with uniparental disomy (UPD), which is an unequal parental contribution to the genome. I have derived mouse embryonic stem (ES) cell sub-lines with maternal UPD (mUPD) for mouse chromosome 6 (MMU6) to characterize regulation and maintenance of imprinted gene expression. The main finding from this study is that maintenance of imprinting in mitotic UPD is extremely variable. Imprint maintenance was shown to vary from gene to gene, and to vary between ES cell lines depending on the mechanism of loss of heterozygosity (LOH) in that cell line. Certain genes analyzed, such as Peg10, Sgce, Peg1, and Mit1 showed abnormal expression in ES cell lines for which they were mUPD. These abnormal expression levels are similar to that observed in ES cells with meiotically-derived full genome mUPD (parthenogenetic ES cells). Imprinted CpG methylation at the Peg1 promoter was found to be abnormal in all sub-lines with mUPD for Peg1. Two cell sub-lines which incurred LOH through mitotic recombination showed hypermethylation of Peg1, consistent with the presence of two maternal alleles. Surprisingly, a cell sub-line which incurred LOH through full chromosome duplication/loss showed hypomethylation of Peg1. The levels of methylation observed in these sub-lines correlates with expression, as the first two sub-lines showed a near-consistent reduction of Peg1, while the latter showed Peg1 levels close to wild-type. Altogether these results suggest that certain imprinted genes, like Peg1 and Peg10, have stricter imprinting maintenance, and as a result show abnormal expression in UPD. This strict imprint maintenance is disrupted, however, in UPD incurred through full chromosome duplication/loss, possibly because of the trisomic intermediate stage which occurs in this mechanism. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Heterozygous mutations in BRCA1 or BRCA2 predispose carriers to an increased risk for breast or ovarian cancer. Both BRCA1 and BRCA2 (BRCA1/2) play an integral role in promoting genomic stability through their respective actions during homologous recombination (HR) mediated repair and stalled replication fork protection from nucleolytic degradation. SMARCAD1 (SD1) is a SWI/SNF chromatin remodeler that has been implicated in promoting long-range end resection and contributes to HR. Using human cell lines, we show that SMARCAD1 promotes nucleolytic degradation in BRCA1/2-deficient cells dependent on its chromatin remodeling activity. Moreover, SMARCAD1 prevents DNA break formation and promotes fork restart at stalled replication forks. These studies identify a new role for SMARCAD1 at the replication fork. In addition to the work presented here, I discuss a method for introducing stop codons (nonsense mutations) into genes using CRISPR-mediated base editing, called iSTOP, and provide an online resource for accessing the sequence of iSTOP sgRNASs (sgSTOPs) for five base editor variants (VQR-BE3, EQR-BE3, VRER-BE3, SaBE3, and SaKKH-BE3) in humans and over 3 million targetable gene coordinates for eight eukaryotic species. Ultimately, with improvements to CRISPR base editors this method can help model and study nonsense mutations in human disease.

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Methods of calculating the inbreeding coefficient In a finite population undergoing recurrent selection (self-select-intercross in succeeding generations) were investigated. It was noted that, in a population under selection, the inbreeding coefficient does not provide the experimenter with a measure of expected degree of variability; instead an index of total heterozygosity is required, and such an Index was derived. Formulas necessary to calculate both the inbreeding coefficients and the heterozygosity indexes were derived for the cases: one-locus, two-allele, random selection; k independent loci and random selection; one-locus, two-allele and effective directional selection; and k linked loci with effective directional selection. These formulas Involved defining a generalized inbreeding coefficient and a generalized index of homozygosity (or heterozygosity) in terms of vectors whose components reflected the various possible patterns of genes identical by descent at a given stage of the recurrent selection breeding program. Formulas were derived whereby the mean and the variance of the total number of loci homozygous (or heterozygous) by descent or in state may be obtained. The progress of the panmictic index and/or the index of total heterozygosity through at least twenty-five cycles of recurrent selection was observed in computer-simulated populations ranging in sizes from ten through one hundred, assuming varying recombination probabilities both in the one-locus and in the two linked-loci case and assuming both minimum and maximum inbreeding selection patterns. Tables resulting from these simulated studies could be used to estimate minimum and maximum inbreeding coefficients and/or minimum and maximum heterozygosity indexes in experimental populations for which the initial conditions approximate those assumed in the simulated populations. It was observed that the coefficient of relationship in the source population was extremely important in tracing the progress of the degree of Inbreeding and/or total homozygosity, that linkage played a major role in promoting heterozygosity in a recurrent selection system, and that careful intercrossing rather than random mating in alternate generations of the recurrent selection cycle was important in promoting maximum heterozygosity in the selected population. In the simulated populations the effect of small population sizes was observed and, in general, indications were that unless more than five complete recurrent cycles are contemplated, increasing population size results In only relatively minor increases in panmixia, especially when linked loci are involved in the selected trait and when care Is taken to avoid a maximum inbreeding selection pattern. / Ph. D.

Thesis: Ph. D., Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Biology; and the Woods Hole Oceanographic Institution), 2017. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Every genome tells a story. This dissertation contains four such stories, focused on shared themes of marine population dynamics and rapid change, with an emphasis on invasive marine species. Biological invasions are often characterized by a range expansion, during which strong genetic drift is hypothesized to result in decreased genetic diversity with increased distance from the center of the historic range, or the point of invasion. In this dissertation, population genetic and genomic tools are used to approach complex and previously intractable fundamental questions pertaining to the non-equilibrium dynamics of species invasions and rapid range expansions in two invasive marine species: the lionfish, Pterois volitans; and the shrimp, Palaemon macrodactylus. Using thousands of loci sequenced with restriction enzyme associated DNA sequencing in these two systems, this research tests theoretical predictions of the genomic signatures of range expansions. Additionally, the first chapter elucidates patterns of population genetic connectivity for deep-sea invertebrates in the New Zealand region demonstrating intimate relationships between genetics, oceanographic currents, and life history traits. Invasive shrimp results extend our understanding of marine population connectivity to suggest that human-mediated dispersal may be as important - if not more important - than oceanographic and life history considerations in determining genetic connectivity during specific phases of marine invasions. In invasive populations of lionfish, measures of genomic diversity, including a difference between observed and expected heterozygosity, were found to correlate with distance from the point of introduction, even in the absence of spatial metapopulation genetic structure. These results indicate a signal of rapid range expansion. The final study in this dissertation uses an innovative temporal approach to explore observed genomic patterns in the lionfish. In all, this dissertation provides a broad perspective through the study of multiple species undergoing superficially parallel processes that, under more intense scrutiny, are found to be mechanistically unique. It is only through comparative approaches that predictable patterns of population dynamics will emerge. / by Eleanor Kathleen Bors. / Ph. D.

Diss. (sammanfattning) Linköping : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.