Assessing hormone receptors (the estrogen and progesterone receptors) and the human epidermal growth factor receptor 2 (HER2) to guide clinical decision making revolutionized treatment for breast cancer patients. However, in the years since these biomarkers were first incorporated into routine clinical care, only a few others have been validated as clinically useful in guiding adjuvant chemotherapy decisions and are recommended by the American Society of Clinical Oncology (ASCO) for patients with hormone-positive breast cancer. For patients with triple-negative breast cancer (TNBC), which lacks hormone and HER2 receptors, not any of these biomarkers are recommended by ASCO due to insufficient evidence that they meaningfully improve clinical outcomes. Breast cancer is the second-leading cause of cancer-related death among women in the US, indicating an unmet need to improve treatments, which can be accomplished in part by identifying and validating novel predictive and prognostic biomarkers that yield actionable information about the clinical course of breast cancers, especially TNBCs. A major obstacle to improving outcomes for breast cancer patients is intratumor heterogeneity (ITH), which can be extensive in breast cancer and drives treatment resistance and relapse. I envision that assaying drivers of ITH can inform clinicians about which breast tumors may be intrinsically more aggressive and carry a greater risk of breast cancer-related morbidity and mortality. My research, presented here, primarily focuses on testing the impact of drivers of ITH (namely, centrosome amplification [CA], the clustering protein KIFC1, and mitotic propensity and its drivers) on clinical outcomes in breast cancer in multivariable models as well as the correlates of in vitro efficacy of centrosome declustering drugs (which can selectively eliminate cancer cells with CA). Collectively, these studies reveal gene signatures and immunohistochemical biomarkers that are independent predictors of aggressive breast cancer course and rational strategies to optimize targeted therapy to combat cancer cells exhibiting CA, thereby contributing to the literature on the development of precision medicine for breast cancer patients, including TNBC patients.

It is known that age-related changes impacting multiple organ systems occur earlier in people with Down syndrome (Ds), but the biological basis underlying this trisomy 21-associated propensity for premature aging is poorly understood. Given that the trisomic/normal cells from people with mosaic Ds (mDs) are identical with regards to environmental exposures and genes (except for chromosome 21 copy number), comparisons of these isogenic trisomic/disomic cells allow one to “unmask” the cellular consequences of trisomy 21 by removing extraneous factors. The primary aim of this study was to determine if trisomy 21 results in an increase in the acquisition of age-related somatic chromosomal changes. To meet this aim, chromosome-specific telomere lengths, senescence-associated distension of satellites (SADS), and chromosomal instability frequencies were compared between the isogenic trisomic/disomic cells of people with mDs ranging from 1 to 44 years of age. Chromosome-specific telomere lengths were quantified using a Q-FISH (pantelomeric probe) method. The average trisomic cell telomere length (3.609 mean, +/- 0.082 SE) was significantly less than the average disomic cell telomere length (3.888 +/- 0.083) (n=28; p

New and more advanced prenatal tests have steadily been introduced in the Swedish maternity care system in the last 30 years. The combined test, CUB, was introduced step wise in Swedish maternal care from 2008 and onward. The CUB test detects children with chromosomal abnormalities prenatally and is offered at no charge for women in treated counties. This thesis investigate the reform using a difference-in-difference approach to determine the effect of the CUB test on the number of late abortions performed. My theoretical framework suggest that the introduction of CUB should increase the number of abortions of children with chromosomal aberrations. As supported by theory I find a positive effect of CUB on late abortions for my main group of interest, women 35-39 years old. These women were the ones most effected by CUB. The positive effect of 0.47 percentage units is statistically significant at the 10% level. It corresponds to a 3.6-7.1% decrease in the number of babies born with chromosomal aberrations.

In order to equally segregate their genetic material into daughter cells during mitosis, it is essential that chromosomes undergo major restructuring to facilitate compaction. However, the process of transforming diffuse, entangled interphase chromatin into discrete, highly organised chromosomal structures is extremely complex, and currently not completely understood. The complexes involved in chromatin compaction and sister chromatid decatenation in preparation for mitosis include condensins I and II. Mutations in condensin subunits have been identified in human tumours, reflecting the importance of accurate cell division in the prevention of aneuploidy and tumour formation. Most mutations described in TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) are missense, and therefore likely to only partially affect condensin function. Most functional genetic studies of condensin, however, have used loss of function systems, which typically cause severe chromosome segregation defects and cell death. Mice carrying global hypomorphic mutations within the kleisin subunit of the condensin II complex develop T cell lymphomas. The Caph2nes/nes mouse model is therefore a good system for understanding how condensin dysfunction can influence tumourigenesis. However, little is known about which cellular processes are affected in mutant cells before transformation. I therefore set out to use the Caph2nes/nes mouse model to study the consequences of the condensin II deficiency on cell cycle regulation in several different hematopoietic lineages. The Caph2nes/nes mice are viable and fertile, with no obvious abnormalities other than the thymus, which is drastically reduced in size. Previous studies reported greater than a hundred-fold reduction in the number of CD4+ CD8+ thymocytes. I set out to understand why the alteration of a ubiquitously expressed protein which functions in a fundamental cellular process would result in such a cell-type specific block in development. To achieve this, I investigated the possibility that condensin II is involved in interphase processes as well as in mitosis. In addition, I studied the aspects of T cell development that may make this lineage particularly vulnerable to condensin II deficiency. Finally, I carried out a preliminary investigation into the biochemical properties of the condensin complexes. During my PhD., I found strong evidence to suggest that the Caph2nes/nes T cell-specific phenotype arises due to abnormal cell division. However, I was unable to find any evidence to support the hypothesis that the phenotype is a consequence of abnormal interphase processes. Upon systematic analysis of several stages of hematopoietic differentiation, I found that at a specific stage of T cell development, the mutation results in an increased proportion of cells with abnormal ploidy, followed by a drastic reduction in cell numbers. Erythroid cells revealed a similar increase in the frequency of hyperdiploid cells, but no reduction in cell numbers. B cells and hematopoietic precursors did not reveal an increase in hyperdiploidy, or a reduction in cell numbers in wildtype relative to mutant. Subsequently, I found preliminary evidence to suggest that the T cell-specificity may be due to more rapid progression of CD4+ CD8+ T cells from S phase to M phase, relative to other hematopoietic stages. Finally, a preliminary investigation into the biochemical properties of the condensin complex revealed apparent imbalances in the expression of condensin subunits in T, B and erythroid cells. The sedimentation profile of CAP-H2 from whole-thymus extract did not exclude the possibility that condensin subunits might be forming heavier-weight complexes with non-SMC proteins. Further work must be carried out to determine whether this sedimentation pattern is unique to T cells.

With 50,310 related deaths this year, colorectal cancer (CRC) has emerged as the second largest cause of cancer related deaths among Americans. While 70 million Americans are considered at-risk of developing CRC, it is highly curable if detected early. Cohesin proteins, which hold sister chromatids together during replication, have emerged as a potential biomarker in multiple cancer lines. Because of their probable role in DNA replication, DNA repair, chromatin nano-architecture, and gene expression, this paper assessed whether cohesion proteins could be used as a potential biomarker for colorectal cancer risk stratification. While cohesin protein mutations have been reported in different cancers and involved in chromosomal instability, its role in early cancer formation has yet to be observed. Using immunohistochemical and Quantitative Real Time PCR analysis, this thesis assessed the protein and RNA expression levels of cohesin proteins SA-1, NIPBL, and SMC3 from human biopsies at different stages and locations of colorectal cancer development. The results showed that SA-1, a structural cohesion subunit, was significantly (p<0.01) down regulated in cancerous compared to normal tissue. The SA-1 protein was also down regulated in the involved mucosa adjacent to CRC polyps. The cohesion loading protein, NIPBL, was also significantly (p<0.01) under expressed in cancerous versus normal tissue. The RNA expression analysis of rectal mucosa showed that SMC3 and SA-1 was over expressed two fold in patients harboring hyperplastic and adenomous polyps, giving evidence that cohesin proteins are differentially expressed throughout the field of carcinogenesis. Our results demonstrate for the first time that cohesion dysregulation is an early event in human colorectal cancer development and may serve as an important biomarker of field carcinogenesis.

In Drosophila oocytes, chromosomes undergo dynamic reorganisation during the prophase of the first meiotic division. This is essential to prepare chromatin for synapsis, recombination and consequent chromosome segregation. The progression of meiotic prophase I is well described, while the molecular mechanisms and regulation of these dramatic chromosomal reorganisations are not well understood. Histone modifying enzymes are major regulators of chromatin structure, however, our knowledge of their roles in meiotic prophase I is still limited. In this work, I investigated the role of the histone demethylase Kdm5/Lid, which removes one of the trimethyl groups at Lys4 of Histone 3 (H3K4me3). I showed that Kdm5/Lid is important for the assembly of the synaptonemal complex, pairing of homologous centromeres, and the karyosome formation. Additionally, Kdm5/Lid promotes crossing over and therefore ensures accurate chromosome segregation. Although loss of Kdm5/Lid dramatically increased the level of H3K4me3 in oocytes, catalytically inactive Kdm5/Lid rescued the above cytological defects. Thereby, I found that Kdm5/Lid regulates chromatin architecture in meiotic prophase I oocytes independently of its demethylase activity. To further identify the regulators of meiotic chromatin organisation during prophase I, I carried out a small-scale RNAi screen for karyosome defects. I found that depletion of ubiquitin ligase components, SkpA, Cul-3 and Ubc-6, disrupted the karyosome formation and the assembly of the synaptonemal complex. The success of the small-scale screen motivated me to initiate the genome-scale RNAi screen for karyosome defects. I found 40 new genes that, when depleted, strongly impaired karyosome morphology. Further studies are required to confirm and elucidate their role in chromatin organisation in oocytes. Overall, my findings have advanced our understanding of the regulation of chromatin reorganisation during oocyte development. Because of the conservation between Drosophila and human meiosis, this study provides novel insights into the regulation of meiotic progression in human oocytes.

The chromosomal passenger complex (CPC: Aurora B-INCENP-Survivin-Borealin) is a key regulator of cell division whose localisation at centromeres is required for stable kinetochore-microtubule attachments and proper chromosomal segregation (Ruchaud et al. 2007; Carmena et al. 2012; van der Waal et al. 2012). Shugoshin1 (hSgo1) (via Borealin) and Histone H3 (via Survivin) have been implicated in centromeric targeting of CPC (Wang et al. 2010; Jeyaprakash et al. 2011; Tsukahara et al. 2010; Kawashima et al. 2010). Although the Survivin-Histone H3 pathway has been extensively studied, the intermolecular interactions dictating CPC-hSgo1 interactions remain unclear. My PhD work focused on characterising the molecular framework of the CPC-hSgo1 interaction using biochemical, biophysical and structural biology methods. I optimised and improved human CPC and hSgo1 recombinant protein production in an E. coli system. Post optimisation, I used Size-Exclusion Chromatography to successfully reconstitute the CPC-hSgo1 complex in vitro and further confirmed that hSgo1 possessing no modification or extra amino acids on its N-terminus can interact with Survivin and Borealin-Survivin-INCENP1-57. This suggested that the hSgo1 N-terminal tail interaction with Survivin is crucial for CPC-hSgo1 interaction. Furthermore, I conducted calorimetric binding studies to molecularly dissect the individual contributions of CPC components and their domains towards CPC-hSgo1 interaction. Towards this aim, I expressed and purified different versions of CPC and analysed their binding energetics with hSgo1. The results from these experiments clearly suggested the contribution of Borealin and INCENP towards CPC-hSgo1 interaction.

Orientador: Humberto Tonhati / Resumo: No primeiro capítulo foi proposto um estudo de associação das informações genômicas com características de tipo, a fim de identificar regiões cromossômicas e genes possivelmente relacionados à altura, peso, crescimento e fatores de conformação corporal. Utilizou-se um painel de 49.010 marcadores SNPs (322 animais) conjuntamente com informações de pedigree (674 animais). As analises foram processadas através do programa Blup.f90, utilizando-se a metodologia de single-step (ssGBLUP). Somente as janelas com 10 SNPs adjacentes e que explicavam acima de 1,5% de variância genética aditiva, foram considerados. Os genes SYT10, GKAP1, C9orf64 e PHLPP1 foram identificados para a característica altura da cernelha (ALTC) e apresentam influência para crescimento, altura e desenvolvimento ósseo. Também foi reportado um QTL para ALTC influenciando crescimento e fertilidade. Foram encontrados SNPs localizados em regiões cromossômicas que ainda não foram previamente descritos com QTL para as características em estudo em búfalos. No segundo capítulo, foram estimados os parâmetros genéticos/genômicos para as características produção de leite acumulada aos 305 dias (PLDC) e caracteristicas de tipo altura da cernelha (ALTC), altura da garupa (ALTG), comprimento do corpo (COMPC), comprimento da garupa (COMPG), Largura entre os íleos (LILE), Largura entre os ísquios (LISQ) e perímetro torácico (PERTOR) em búfalos da raça Murrah utilizando modelos bicaracterísticos. As estimativas de herdabilidade f... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In the first chapter we proposed a study of the association of genomic information with type characteristics, in order to identify chromosomal regions and genes possibly related to height, weight, growth and body conformation factors. Were used one panel of 49,010 SNPs markers (322 animals) together with pedigree information (674 animals). The analyzes were processed through the Blupf90 program, using the singlestep methodology (ssGBLUP). Only the windows with 10 adjacent SNPs and, that explained above 1.5% of additive genetic variance, were considered. The genes SYT10, GKAP1, C9orf64 and PHLPP1 were identified for the characteristic height of the withers (ALTC) and presented influence on growth height and bone development. It has also been reported a QTL for ALTC influencing growth and fertility. SNPs were found located in chromosomal regions which have not previously been described with QTL for the characteristics under study in buffaloes. In the second chapter, were estimated the genetic / genomic parameters for the characteristics accumulated milk production at 305 days (AMP) and characteristics of the withers height (WH), croup height (CH), body length (BL), croup length (CL), Width between hip bones (WBHB), Width between pin bones (WBPB) and thoracic perimeter (TP) in Murrah buffaloes using characteristic models. Genetic correlations were 0.603 between AMP and WH, 0.898 between WBHB and BL, 0.867 betwwen AMP and CL, 0.887 between AMP and TP, 0.774 between WBHB and WBPB,... (Complete abstract click electronic access below) / Mestre

Natural environments expose organisms to multifarious selective pressures involving numerous aspects of the overall phenotype, therefore eliciting a response from multiple correlated loci. It has been hypothesized that chromosomal rearrangements can play a role in facilitating local adaptation by establishing new linkage relationships and modifying the recombination patterns between the different chromosomal forms, allowing coordinated adaptation of several loci. The central aim of the work presented here is to test this hypothesis using Drosophila americana as a model system. This species segregates several inversions and an X-4 centromeric fusion which makes it an excellent model to study the role of chromosomal rearrangements on local adaptation. This hypothesis was tested using several approaches. The geographic distribution of the chromosomal rearrangements was determined through sampling of wild populations from a broad geographic range. It was found that several of the chromosomal rearrangements exhibit clinal variation. Furthermore, many of these are found in high linkage disequilibrium. The X-4 fusion is highly associated with inversions on the X and 4th chromosome. Also, two inversions on chromosome 5 are in strong negative linkage disequilibrium. The sequence variation associated with rearrangements of the X was studied using inbred lines. The results show that the inversion and the fusion strongly influence variation on this chromosome. Regions of significant population differentiation and linkage with the rearrangements are found interspersed with loci showing neutral variation indicating that in spite of recombination, allelic associations are maintained on this chromosome. The analysis was also extended to flies directly collected from the wild sampled from a region encompassing a large part of the species' range. Loci throughout chromosome X and 4 were genotyped. Sites in high linkage disequilibrium with the rearrangements and with other assayed sites were found in close proximity with sites that did not show this pattern. In conclusion, the clinal distribution of chromosomal rearrangements and associated genetic variation in conjunction with the detection of islands of linkage disequilibrium among the rearrangements and loci on both chromosomes indicate that chromosomal rearrangements are facilitating local adaptation in D. americana.

Two manifestations of gene regulation-- dosage compensation and ontogenic regulation--were examined in normally positioned and relocated v+ genotypes in Drosophila melanogaster to determine the role of gene position in these control functions. Enzyme assays, used as criteria of gene activity, were performed on various genotypes containing different doses of v+ in normal and relocated positions in male and female flies. The results indicate that although differently positioned v+ genes may specify different tryptophan pyrrolase activities, they still show dosage compensation. In each case, the enzyme activity associated with each gene, either on the X, Y, or third chromosome, is twice as much in males as it is in females. This indicates that dosage compensation is not confined to the gene when located on the X chromosome. In order to determine if the pattern of activity of the gene during ontogeny is altered by relocation, T(l; 3)rasv genotypes and wild type controls were assayed at the same stages of development. The experimental design allowed a comparison of the ontogenic expression of three different genes--v+, Zw, and Pgd--through the activities of their associated enzymes. The results indicate that changing the gene's position may alter its ontogenic expression. Animals with v+ on the third chromosome have a unique peak of tryptophan pyrrolase activity in larvae which is not present in wild type. The activity in this peak is at l east 10 times higher than that observed in 72-hour wild type larvae, in fact, higher than that observed in any normal genotype at any time during development. With the exception of this peak, the developmental curves of enzyme activity are similar, although the relocated genes specify consistently lower enzyme activities than do normally positioned genes. The unique peak is not the result of a general physiological effect since the patterns of Zw and Pgd activity appear to be the same in wild type and translocated v+ genotypes. The relevance of the data to earlier studies and to models for gene regulation is discussed.