Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 163-179).

<p>Every living cell faces a multitude of DNA threats in its lifetime because damage to DNA is intrinsic to life itself. A double-strand break (DSB) is the most cytotoxic type of DNA damage and is a potent inducer of chromosomal aberrations. Defects in DSB repair are a major driver of tumorigenesis and are associated with numerous developmental, neurological and immunological disorders. To counteract the deleterious effects of DSBs, organisms have evolved a homologous repair (HR) mechanism that is highly precise. The key to its error-free nature lies in its use of a homologous template in restoring the DSB and its preferential occurrence during late S and G2 phase of the cell cycle when identical sister chromatids are available as templates for repair. However, HR can also engage homologous chromosomes and ectopic substrates that share homology, resulting in mitotic loss-of-heterozygosity (LOH) and unwanted chromosomal aberrations. In this case, understanding of the underlying mechanisms and molecular factors that influence accurate sequence transfer and exchange between two homologous substrates becomes crucial. </p><p>The focus of this dissertation is examination of the genetic factors and molecular processes occurring at early intermediate steps (DNA end resection and DNA synthesis) of mitotic recombination in Saccharomyces cerevisiae. To model DSB repair, we established a unique plasmid-based assay with a small 8-base pair (bp) gap in the middle of an 800-bp plasmid substrate. To delineate the molecular structures of strand exchange intermediates during HR, we used a 2% diverged plasmid substrate relative to a chromosomal repair template to generate mismatch-containing heteroduplex DNA (hetDNA) intermediates. The assay was performed in a mismatch repair (MMR)-defective background allowing hetDNA to persist and to segregate into daughter cells at the next round of replication. Unexpectedly, even when MMR was inactivated, sequence analysis of the recombinants revealed patches of gene conversion and restoration reflecting mismatch correction within hetDNA tracts. We showed that, in this system, MMR and nucleotide excision repair (NER) correct mismatches via two different mechanisms. While mispairing of nucleotides triggers MMR, NER is recruited by the subtle 6-methyladenine mark on the plasmid substrate, leading to coincident correction of mismatches. The methylation marks on the plasmid were acquired from the bacterial host’s native restriction-modification system during plasmid propagation. </p><p>Formation of hetDNA occurs when a plasmid substrate engages the chromosomal template for repair, forming a D-loop intermediate. D-loop extension requires DNA synthesis by DNA polymerase/s. Translesion synthesis (TLS) polymerases have been implicated in HR in both chicken DT40 cells and fruit fly, but not in yeast. This class of polymerases is known for its low fidelity due to a lack of exonuclease domain and is commonly used for lesion bypass and in extending ends with mismatches. We reported for the first time a requirement of Polζ-Rev1 and Polη (TLS polymerases in S. cerevisiae) for completing gap repair. Moreover, gap-repair efficiency suggested that these two polymerases function independently. We concluded that TLS polymerases are involved in either extending the invading 3’ end and/or in the gap-filling process that completes recombination. </p><p>DNA resection of a DSB serves as a primary step to generate a 3’ single-stranded DNA (ssDNA) for subsequent homologous template invasion, but this process has mostly been studied in the absence of a repair template or when downstream HR steps are disabled. To analyze the individual contributions of identified nucleases to DSB resection in the context of repair, we established a chromosomal assay; the substrate size was increased to 4 kilobases (kb) and 85 SNPs were present at ~50 bp intervals. In this chromosomal assay, resection and DNA synthesis influence the length of hetDNA tracts in the final recombinants, allowing these two steps to be analyzed. We specifically focused on synthesis-dependent strand annealing (SDSA) events, where hetDNA reflects DNA synthesis and extent of resection. Our main conclusions are as follows. DNA end resection on the annealing end of NCO products generated by SDSA is not as extensive as one might expect from resection measured in single-strand annealing (SSA) assays. In addition, although the two long-range resection pathways (Sgs1-Dna2 and Exo1) can support recombination in a redundant manner, hetDNA was significantly reduced upon loss of either. End processing of DSBs is predominantly 5’ to 3’, but we also observed loss of sequences (greater than 8 nt but less than 40 nt) at the 3’ termini. We have tested and ruled out the involvement of Mre11 and Polε proofreading activity. Lastly, Pol32 functions as a subunit of Polδ to promote extensive repair synthesis during SDSA. hetDNA tract lengths were significantly shorter in the absence of the Pol32 subunit of Polδ, providing direct evidence that Polδ extends the invading end during HR. Together, this work advances our understanding of how resection nucleases and DNA polymerase/s function to regulate mitotic recombination outcome and influence the molecular patterns of NCOs.</p> / Dissertation

Common fragile sites are a set of genomic locations with a propensity to form lesions, breaks and gaps on mitotic chromosomes upon induction of replication stress. While the exact reasons for their fragility are unknown, CFS display instability in a cell-type specific manner, suggesting a substantial contribution from an epigenetic component. CFSs also overlap with sites of increased breakage and deletions in tumour cells, as well as evolutionary breakpoints, implying that their features shape genome stability in vivo. Previously, factors such as delays in replication timing, low origin density and transcription of long genes have been implicated in instability at CFS locations but comprehensive molecular studies are lacking. Chromatin structure, an important factor that fits the profile of cell-type specific contributor, has also not been investigated yet. Throughout their efforts to determine the factors that lead to the appearance of CFS lesions, investigators have focused on a single component at a time, potentially missing out complex interactions between cellular processes that could underlie fragility. Additional difficulties come from the cell-type specificity of CFS breakage: it indicates that only cell type-matched data would be informative, limiting the scope for studies using publicly available data. To perform a comprehensive study defining the role of different factors in determining CFS fragility, I explored replication timing, transcriptional landscapes and chromatin environment across a number of CFSs in two cell types exhibiting differential CFS breakage. Initially, I characterised the patterns of CFS fragility in the two cell types on both the cytogenetic and the molecular level. I then used a FISH-based technique to investigate the process of mitotic compaction at active CFS sites and found that the cytogenetically fragile core of these sites sits within larger regions which display a tendency to mis-fold in mitosis. The aberrant compaction of these regions could be observed on cytogenetically normal metaphase chromosomes, suggesting that finer scale abnormalities in chromosome structure underlie the cytogenetically visible breaks at fragile sites. I also investigated the links between transcription of long genes and CFS fragility using two approaches: I quantified levels of expression across all fragile sites using RNA-seq and modified transcription at a single active CFS using the CRISPR genome engineering methodology. My results indicate a complex interplay between transcription and CFS fragility: no simple linear correlation can be observed, but an increase of transcriptional levels at the active CFS led to a corresponding increase in fragility. To investigate the influence of the cell type specific replication programme and replication stress on CFS instability, I mapped replication timing genome-wide in unperturbed cells and under conditions of replication stress in both cell types. I found that replication stress induces bi-directional changes in replication timing throughout the genome as well as at CFS regions. Surprisingly, the genomic regions showing the most extreme replication timing alterations under replication stress do not overlap with CFS, implying that CFS instability is not fully explained by replication delays as previously suggested. Instead, I observed a range of replication-stress induced timing changes across CFS regions: while some CFSs appear under-replicated, others display switches to both earlier and later replication as well as differential recruitment of both early and late origins, implying that dis-regulation of replication timing and origin firing, rather than simply delays, underlie the sensitivity to CFS regions to replication stress. Finally, I investigated large-scale chromatin states at two active CFSs throughout S phase and into G2, the cell cycle stages most relevant stage for CFS breakage. I found that changes in large-scale chromatin architecture accompany the replication timing shifts triggered by replication stress, raising the possibility that such alterations contribute to instability. In conclusion, I assessed the influence of multiple relevant factors on CFS fragility. I found that bi-directional replication timing changes and alterations in interphase chromatin structure are likely to play a role, converging to promote mitotic folding problems which ultimately result in the well-described cytogenetic lesions on metaphase chromosomes and genomic instability.

Proper orientation of the mitotic spindle is essential during animal development for the generation of cell diversity and organogenesis. To understand the molecular mechanisms regulating this process, genetic studies have implicated evolutionarily conserved proteins that function in diverse cell types to align the spindle along an intrinsic cellular polarity axis. This activity is achieved through physical contacts between astral microtubules of the spindle and a distinct domain of force generating proteins on the cell cortex. In this work, I shed light on how these proteins form distinct cortical domains, how their activity is coupled to their subcellular localization, and how they provide cytoskeletal and motor protein connections that are required to generate the forces necessary to position the mitotic spindle. I first discuss the mechanisms by which Mushroom body defect (Mud; NuMA in mammals), provides spindle orientation cues from various subcellular locations. Aside from its known role at the cortex as an adapter for the Dynein motor, I reveal novel isoform-dependent Mud functions at the spindle poles during assembly of the mitotic spindle and astral microtubules, thus implicating Mud in spindle orientation pathways away from the cell cortex. Moreover, through collaborative efforts with former lab members, I describe molecular regulation and assembly of two ‘accessory’ pathways that activate cortical Mud-Dynein, one through the tumor suppressor protein Discs large (Dlg), and another through the signaling protein Dishevelled (Dsh). I demonstrate that the Dlg pathway is spatially regulated by the polarity kinase atypical Protein Kinase C (aPKC) through direct phosphorylation of Dlg. This signal relieves Dlg autoinhibition to promote cortical recruitment of the Dlg-ligand Gukholder (Gukh), a novel microtubule-binding protein that provides an additional connection between astral microtubules and the cortex that is essential for activity of the Dlg pathway. Lastly, I determine that the Dsh accessory pathway provides an alternative cytoskeletal cue by recruiting Diaphanous (Dia), an actin nucleating protein. By demonstrating interchangeability between the two accessory pathways, we conclude that Mud-Dynein is activated by various cytoskeletal cues and that the mode of activation is cell-context dependent. This dissertation includes unpublished and previously published co-authored material. / 10000-01-01

Orientador: Júlio Lopes Sequeira / Resumo: As neoplasias mamárias das gatas frequentemente são malignas e agressivas, sendo os tipos mais comuns classificados como carcinomas tubulopapilíferos, sólidos e cribriformes. O grau histológico tem relação com o comportamento biológico desses tumores. No entanto poucos estudos tem abordado a cinética celular, a expressão de marcadores epiteliais e mioepiteliais, ou mesmo de moléculas de adesão e suas relações com a agressividade tumoral. Assim, o objetivo deste trabalho foi relacionar o tipo histológico dos carcinomas mamários das gatas e seus graus histológicos com os índices proliferativos e apoptóticos, e a expressão imuno-histoquímica de CK14, alfa-SMA, E-caderina e P-caderina. Foram utilizadas 31 amostras de carcinomas mamários de gatas. Submetidas a técnica imuno-histoquímica indireta com os anticorpos Ki-67, caspase-3-clivada, CK14, alfa-SMA, E-caderina e P-caderina. Predominaram as gatas SRD, com média de idade de 12 anos. Em frequência, o percentual dos tipos histológicos foi: 42%, 45,50% e 12,50% para os carcinomas tubulopapilíferos, sólidos e cribriforme; e foi de 9,65%, 41,95% e 48,80%, para os graus I, II e III, respectivamente. Os carcinomas tubulopapilíferos mostraram índice mitótico inferior aos carcinomas sólidos, assim como os carcinomas de grau I em relação aos de grau II e III. A característica basal (CK14 +) foi frequente nesses carcinomas. O subtipo complexo (alfa-SMA + ou alfa-SMA/CK14 +/-) é raro. Houve perda da expressão de E-caderina a medida que se ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Mammary tumors of the cats are often malignant and aggressive, the most common types classified as tubulopapillary, solid and cribriform carcinomas. The histological grade is related to the biological behavior of these tumors. However few studies have addressed the cell kinetics, the expression of epithelial and myoepithelial markers, or adhesion molecules and their relationship with tumor aggressiveness. The objective of this study was to correlate the histologic type of breast carcinomas of the cats and their histological grades with proliferative and apoptotic indices, and immunohistochemical expression of CK14, alpha-SMA, E-cadherin and Pcadherin. 31 samples of breast carcinomas cats were used. Subjected to indirect immunohistochemical technique with antibodies Ki-67, caspase-3, cleaved CK14, alpha-SMA, E-cadherin and P-cadherin. Predominated SRD cats, with a mean age of 12 years. In frequency, the percentage of histological types was 42%, 45.50% and 12.50% for tubulopapillary, solid and cribriform carcinomas; and it was 9.65%, 41.95% and 48.80% for grades I, II and III, respectively. The tubulopapillary carcinomas showed mitotic index lower than the solid carcinomas, as well as grade I carcinomas compared to levels II and III. The basal feature (CK14 +) was common in these carcinomas. The complex subtype (alpha-SMA + or alpha-SMA / CK14 +/-) is rare. There was a loss of Ecadherin expression as it becomes more aggressive. The P-cadherin was expressed high regardless of hi... (Complete abstract click electronic access below) / Mestre

abstract: In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, first, the co-localization and close molecular proximity of PKC isoforms with GSK3â was studied in metaphase cells. Second, the involvement of inactive GSK3â in maintaining an intact mitotic spindle was shown. Third, this study showed that addition of a phospho-PKCæ specific inhibitor to cells can disrupt the mitotic spindle microtubules. The mitotic spindle at metaphase in mouse fibroblasts appears to be maintained by PKCæ acting through GSK3â. The MAPK pathway has been implicated in various functions related to cell cycle regulation. MAPKK (MEK) is part of this pathway and the extracellular regulated kinase (ERK) is its known downstream target. GSK3â and PKCæ also have been implicated in cell cycle regulation. In the study in chapter 3, we tested the effects of inhibiting MEK on the activities of ERK, GSK3â, PKCæ, and á-tubulin. Results from this study indicate that inhibition of MEK did not inhibit GSK3â and PKCæ enrichment at the centrosomes. However, the mitotic spindle showed a reduction in the pixel intensity of microtubules and also a reduction in the number of cells in each of the M-phase stages. A peptide activation inhibitor of ERK was also used. Our results indicated a decrease in mitotic spindle microtubules and an absence of cells in most of the M-phase stages. GSK3â and PKCæ enrichment were however not inhibited at the centrosomes. Taken together, the kinases GSK3â and PKCæ may not function as a part of the MAPK pathway to regulate the mitotic spindle. / Dissertation/Thesis / Ph.D. Molecular and Cellular Biology 2012

Orientador: Gonçalo Amarante Guimarães Pereira, Juan Lucas Argueso Gomes de Almeida / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T13:31:37Z (GMT). No. of bitstreams: 1 Duarte_FabianadeMelo_M.pdf: 10932684 bytes, checksum: c31d50d399af1063941a0d931eee8101 (MD5) Previous issue date: 2010 / Resumo: O etanol de cana-de-açúcar brasileiro ocupa um lugar de destaque entre as alternativas energéticas disponíveis atualmente. No processo fermentativo de produção de etanol é utilizada a levedura Saccharomyces cerevisiae, com destaque para a linhagem industrial PE-2, utilizada por cerca de 30% das usinas brasileiras, o que representa 10% da produção mundial. Essa linhagem associa uma alta eficiência na produção de etanol com uma excelente capacidade de adaptação ao ambiente altamente hostil e competitivo das dornas de fermentação. O nosso grupo de pesquisa realizou previamente uma caracterização genética e molecular detalhada de uma linhagem diplóide derivada diretamente de PE-2 (JAY270), o que forneceu inúmeras possibilidades para a manipulação genética dessa levedura com o objetivo de desenvolver linhagens mais produtivas. No entanto, alguns estudos observaram a ocorrência de variação cariotípica nessa linhagem durante o processo fermentativo, o que pode representar uma barreira para a manipulação dessa levedura. Sendo assim, é extremamente importante estudar o comportamento do genoma dessa linhagem durante a produção de etanol. Este projeto de Mestrado teve como objetivo principal a determinação do mecanismo de recombinação genética responsável pela geração de rearranjos cromossômicos na linhagem JAY270 durante a produção de etanol. Foi realizado um experimento de fermentação em escala semi-industrial iniciado com um inóculo puro de JAY270, com duração de 50 dias. A partir de amostras coletadas ao final do processo foram obtidos isolados que foram analisados através de PFGE (Gel de Eletroforese em Campo Pulsado) para identificação de derivados de JAY270 portadores de variação cariotípica. Dos derivados analisados, 36% possuíam algum tipo de rearranjo cromossômico em relação à linhagem inicial, dos quais 11 foram selecionados (FDY1-FDY11) para análises detalhadas. As sequências genômicas de dois derivados haplóides de JAY270 possibilitaram o desenvolvimento de 9 marcadores moleculares para genotipar regiões heterozigotas de JAY270 com o objetivo de identificar eventos de recombinação genética. Os 11 isolados selecionados foram analisados com esses marcadores e, exceto por um deles, todos continuavam heterozigotos em todas as regiões genotipadas. Dois mecanismos de recombinação genética podem ser responsáveis pela geração dos rearranjos, recombinação mitótica, que ocorre em pontos isolados e recombinação meiótica, que envolve todo o genoma simultaneamente em um único ciclo celular. A possibilidade dos rearranjos terem sido gerados por recombinação meiótica foi excluída, visto que a probabilidade dos 10 isolados continuarem heterozigotos em todas as regiões analisadas após um evento de meiose seguido de cruzamento é extremamente baixa (entre 10-24 e 10-14). Sendo assim, concluiu-se que os rearranjos cromossômicos foram gerados durante o crescimento vegetativo, e que, portanto, o mecanismo de recombinação genética responsável pela geração dos mesmos é a recombinação mitótica. Os 11 isolados também foram analisados através de CGH-array, que possibilitou a comparação de sua dosagem gênica com a da linhagem JAY270, que lhes deu origem. Com essa metodologia foi possível detectar eventos de recombinação mitótica que resultaram em perda de heterozigosidade nas extremidades de alguns cromossomos. / Abstract: Brazilian sugar cane ethanol stands out among the energetic alternatives available nowadays. In the fermentation process to produce ethanol it is used the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is the industrial isolate PE-2, currently used by ~30% of Brazilian distilleries, generating ~10% of the world's ethanol supply. This strain combines a high performance in ethanol fermentation and an excellent adaptation to the hostile environment in the fermentation vats. Our research group previously carried out a thorough genetic characterization of an isolate of the PE-2 strain, known as JAY270. This analysis offers several opportunities for the improvement and modification of this strain. However, some studies observed the occurrence of karyotypic variation in this strain during the fermentation process, what can represent a barrier to the genetic manipulation of this yeast. Therefore, it is extremely important to study the genome behavior of this yeast during the ethanol production process. The main objective of this Master's Project was to determinate the genetic recombination mechanism responsible for generating chromosomal rearrangements in JAY270 during the ethanol production. A fermentation experiment in semi-industrial scale was carried out with a pure initial inoculum of JAY270. After 50 days, samples were collected and used to obtain single colony isolates. This isolates were analyzed through PFGE (Pulsed Field Gel Electrophoresis) to the identification of JAY270 derivatives carrying karyotypic variation. Of the analyzed derivatives, 36% had at least one chromosomal rearrangement. 11 of these derivatives were chosen to perform detailed analyses and were called FDY1-FDY11. The genome sequences of two haploid derivatives of JAY270 were used to develop 9 molecular markers to genotype heterozygous regions of JAY270 to identify genetic recombination events. The 11 isolates were analyzed with these markers and, except by one of them, all isolates were heterozygous in all genotyped regions. Two genetic recombination mechanisms can be responsible for generating the rearrangements, mitotic recombination, which occurs in isolate points of the genome, and meiotic recombination, that simultaneously affects all the genome in one single cellular cycle. The probability of the 10 isolates stay heterozygous in all genotyped regions after one event of meiosis followed by mating is extremely low (between 10-24 e 10-14). This result showed that these rearrangements couldn't be generated through meiotic recombination. Therefore, it was possible to conclude that the chromosomal rearrangements were generated during vegetative growth through mitotic recombination. The 11 isolates also were analyzed through CGH-array to compare their gene dosage with the strain JAY270. With this methodology it was possible to detect mitotic recombination events that resulted in loss of heterozygosity in peripheral regions of some chromosomes. / Mestrado / Genetica de Microorganismos / Mestre em Genética e Biologia Molecular

Os tumores astrocíticos são originados a partir dos astrócitos e classificados de acordo com a Organização Mundial de Saúde em astrocitoma pilocítico (grau I), astrocitoma subependimal de células gigantes (grau I), xantoastrocitoma pleomórfico (grau II), astrocitoma difuso (grau II), astrocitoma anaplásico (grau III) e glioblastoma (GBM) (grau IV). Este último é o tumor cerebral mais frequente em adultos, possuindo uns dos piores prognósticos, devido principalmente à radioresistência do tumor, com uma sobrevida média de 14 meses. Vários estudos têm procurado encontrar novos alvos terapêuticos e os genes da família BUB são candidatos promissores, devido ao seu papel no controle do ciclo celular. Estes genes participam do mecanismo do ponto de checagem do fuso mitótico, prevenindo a separação prematura das cromátides irmãs. Os objetivos deste trabalho foram avaliar a expressão de BUB1, BUB3 e BUBR1 em amostras de pacientes portadores de gliomas de baixo grau (I e II) e glioblastoma, relacionar as expressões destes genes à sobrevida e estudar os efeitos da inibição dos genes BUB1 e BUBR1, por RNAi, na linhagem pediátrica SF188. Nossos resultados mostraram que tanto BUB1 quanto BUBR1 foram hiperexpressos nos glioblastomas e nos gliomas de baixo grau em relação à substância branca (p<0,05). A análise da expressão destes genes em amostras de glioblastomas em relação a amostras de grau I e II demonstrou uma hiperexpressão dos genes BUB1 e BUBR1 e uma hipoexpressão do BUB3 em glioblastoma (p<0,05). Em relação à sobrevida, os baixos níveis de expressão dos genes BUB1 e BUBR1 foram relacionados a uma melhor sobrevida quando analisado o total de todos os pacientes. Paralelamente, a inibição dos genes BUB1 e BUBR1 na linhagem SF188 resultou em uma diminuição da proliferação e também da capacidade clonogênica, além de um aumento na apoptose quando combinado com temozolomida (TMZ). Também foram realizados experimentos com inibições dos genes com ou sem a presença de irradiação. Esta combinação resultou em uma diminuição tanto da proliferação quanto da capacidade clonogênica. Estes resultados sugerem que o BUB1 e o BUBR1 podem ser considerados alvos interessantes para o tratamento de glioblastoma, porém estudos adicionais são necessários para confirmar tal potencial. / Astrocytic tumors are originated from astrocitytes and classified according to the World Health Organization into pilocytic astrocytoma (grade I), subependymal giant cell astrocytoma (grade I), pleomorphic xanthoastrocytoma (grade II), diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and Glioblastoma (GBM) (grade IV). Glioblastoma is the most common brain tumor in adults with a very poor prognosis and a median survival of 14 months. Because their role in cell cycle control, several authors have previously ponited the BUB gene family as new therapeutic target candidates. These genes participate in the mitotic checkpoint by preventing the premature separation of sister chromatids. The aims of this study were to study the expression of BUB1, BUB3 e BUBR1 in samples of patients with low-grade gliomas (I and II) and glioblastoma, evaluate any association of gene expression with patient survival and analyze BUB1 e BUBR1 inhibition (by iRNA) effects, on SF188, a pediatric cell line. BUB1 and BUBR1 were found hyperexpressed in glioblastoma samples and in low-grade gliomas when compared with white matter samples (p<0.05). The analysis of the expression of these genes in glioblastoma samples compared with grade I and II samples showed a hyperexpression of BUB1 and BUBR1, while BUB3 was hipoexpressed in glioblastoma (p<0.05). In the survival analysis, the hipoexpression of BUB1 and BUBR1 genes were related with a better survival when all patients were considered. The BUB1 and BUBR1 inhibition resulted in decreased proliferation and colony formation, along with increased apoptosis when combined with temozolomide. Experiments with the inhibition of genes also were performed in association with irradiation. This combination demonstrated decreased proliferation and colony formation. Our results suggest that BUB1 and BUBR1 might be interesting targets for future treatment of glioblastoma, nonetheless further studies are necessary to confirm this potencial.

A pesquisa de linhagens com duplicação cromossômica, como a linhagem A de Aspergillus nidulans, teve oseu início no final da década de 1970. Durante este período foram isolados da linhagem A, diversos variantes deteriorados, que foram caracterizados genética e citologicamente. Neste trabalho de pesquisa, as analises genéticas demonstraram que os determinantes de deterioração ou segmentos de inserção de V5, V101, V102, V103 e V104 estão localizados nos grupos de ligação VIII, III, IV, VII e I respectivamente. As análises citogenéticas revelaram diversas alterações no ciclo celular e migração nucleares nas fases iniciais de desenvolvimento. A duplicação cromossômica da linhagem A e os variantes deteriorados foram investigados a nível molecular, por técnica de PCR. Os resultados mostraram que o segmento de inserção consiste de um provável Elemento de Transposição, denominado de MATE, o qual é característico do fungo Aspergillus nidulans. Os segmentos de inserção analisados apresentam características típicas de MATE, como o motivo “Spe” que é encontrado por toda seqüência dos Elementos MATE. / The research with chromosome duplication strains, as strain A of Aspergillus nidulans, began during the 70's, with isolation of several deteriorates variants of strain A and characterization by genetic and cytological analysis. In this work the genetic analysis has demonstrated that the deterioration determinant or insertion sequence in V5, V101, V102, V103 and V104 deteriorates variants are located in the linkages groups VIII, III, IV, VII and I, respectively. The cytological analyses have demonstrated changes in cellular cycle and nuclear migration in initial phases of development. The chromosome duplication of strain A and the deteriorated variants were investigated by PCR with designed primers to mobile elements, what have resulted in the identification of the transposable element MATE, mainly by great similarity with "Spe" motif sequence that is described as essential in activity of these elements.

Macrophage-conditioned medium (MacCM) inhibits the differentiation of rodent and human preadipocytes. Previous studies report that murine J774A.1-MacCM inhibits clonal expansion (early required phase of adipogenesis), including Rb phosphorylation. I hypothesized that MacCM induced alterations in cyclins and/or cyclin-dependent kinases (CDKs) were responsible for impairing Rb phosphorylation. My first objective was to assess the effect of J774A.1-MacCM on CDK4, CDK2, and their regulatory cyclins. Murine 3T3-L1 preadipocytes were differentiated with control medium or J774A.1-MacCM. Expression of cyclin D and A was inhibited by J774A.1-MacCM. Inhibition of cyclin A expression was associated with reduced differentiation-induced CDK2 activity. My second objective was to assess the expression patterns of cell cycle proteins in differentiating human abdominal subcutaneous preadipocytes, which do not undergo clonal expansion in culture. Cyclin E expression increased with differentiation. THP-1-MacCM (a human macrophage cell line) further enhanced this increase. My studies suggest MacCM leads to alterations in cyclin/CDK regulation during adipogenesis in murine and human preadipocyte models.