To better understand the evolutionary dynamics of chromosomal inversions, a physical map for an Asian malaria vector, Anopheles stephensi, was created and compared with the maps of the major African malaria vectors A. gambiae and A. funestus No interchromosomal transposition was observed between A. gambiae and A. stephensi. Several cases of euchromatin and heterochromatin transitions weridentified between A. gambiae and A. stephensi. The study of paracentric inversions between lineages in Anopheles mosquitoes demonstrated that X chromosome has the fastest rate of inversion fixations and highest density of repetitive elements. Among the autosomes, 2R evolved faster than other autosomes. The slowly evolved autosomes have more M/SARs than rapidly evolving arms. Breakpoint regions are enriched with repetitive elements. The study revealed that fixed inversions are distributed nonrandomly and breakpoint clustering is common in lineages of A. gambiae and A. stephensi. The parallel association between the density of inversion fixations and polymorphisms suggests that polymorphic inversions can be fixed during evolution. To understand the direction of evolution in A. gambiae complex, the ancestral status of fixed inversions for this complex was identified. The presence of the 2La inversion in outgroups, A. stephensi and A. nili, confirmed the ancestral status of the 2La inversion. The presences of breakpoint structure of the 2Ro inversion in outgroup species, A. stephensi, indicated that the 2Ro is ancestral arrangement. The presence of SINE elements at the breakpoints of the 2R+p in A. gambiae PEST strain suggested that the 2R+p is a derived arrangement. Therefore, the carrier of 2Rop inversions, A. merus, was considered closest to the ancestral species. We have developed a new protocol for laser microdissection and whole genome amplification of polytene chromosomal fragments to obtain DNA for sequencing and assembly. The chromosomal regions spanning both breakpoints of the 2La in A. arabiensis and A. merus were laser microdissected from the polytene chromosomes. Subsequently, DNA samples were amplified using Illustra GenomePhi V2 DNA and Whole-pool amplification methods for obtaining amplicons. Successful amplification of our target DNA was confirmed by PCR with specific primers followed by Sanger sequencing. / Ph. D.

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Ph. D., Faculty of Health Sciences, University of the Witwatersrand, 2008. / The human chromosome complement contains nonrandom genomic regions that are prone to breaks and recurrently altered in tumors. These chromosomal “hot spots” are preferentially involved in early events of genomic instability and point to genes in their vicinity that may participate to carcinogenesis. Amplicons and deletions are frequently generated at “hot spots”, including common fragile sites (CFS), and are thought to host cancer genes whose rearrangements drive cell proliferation and promote the initiation and progression of cancer. Chromosomal “hot spots” in South African cancer patients, were investigated in this study with a view to characterizing underlying gene alterations. A chromosome 12p amplicon was mapped and array comparative genomic hybridization (aCGH) was pioneered to identify candidate genes in the amplicon. Genes of the 12p stem cell gene cluster (NANOG, STELLAR and GDF3) were involved in striking similarity to what has been reported in testicular germ cell tumors and suggest that they may be more commonly involved in different types of cancer. Two tumor suppressor genes, FHIT and WWOX, are located at the two most commonly expressed fragile sites, FRA3B and FRA16D respectively. Alterations in these fragile site associated genes have been reported in a variety of tumors including lung, esophageal, gastric, breast and cervical cancers most frequently as a result of submicroscopic deletions. Genomic deletions at CFS have been mostly investigated using loss of heterozygosity assays that do not necessarily inform on gene exon deletions. A new method was developed based on multiplex ligation-dependent probe amplification (MLPA) that screens for exon deletions/amplifications of genes at CFS. The assay was validated on five esophageal squamous carcinoma cell lines and showed deletions in the FRA3B-associated gene FHIT in four of the cell lines. Two geographically distinct South African cohorts of esophageal squamous cell carcinoma were then screened for FHIT/WWOX exon deletions and a visual basic (VBA) encoded program was written to automate MPLA products analysis. A high frequency of intragenic deletions in FHIT and/or WWOX (73%) was observed in the Eastern Cape cohort. FHIT deletions were seen in 27% of specimens from the Gauteng cohort, which by contrast did not show WWOX deletions. This difference may however reflect a difference in sampling collection. The breakpoints of a translocation t(3;11)(p14;p15.1) present in an ovarian carcinoma cell line was characterized using the above MLPA assay, aCGH and the polymerase chain reaction. The translocation was found to interrupt the FHIT gene making it the 5th cancer associated translocation involving FHIT. The evaluation of gene relative copy number by aCGH and MLPA were highly correlated further validating the power of the MLPA assay in fresh tissue. The involvement of critical genes at “hot spots” in SA cancer patients was high in the context of this study raising questions about the possible role of environmental exposure. The new MLPA assay may assist to expand the screening of critical genes at fragile sites in the future.

Graduation date: 1999

abstract / Pathology / Doctoral / Doctor of Philosophy

Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-09Bitstream added on 2014-06-13T18:29:20Z : No. of bitstreams: 1 coelho_mm_me_botib.pdf: 1279286 bytes, checksum: c9db6d651dfd0584937d8d5948869e47 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Os tumores de glândulas salivares (TGS) são lesões raras que apresentam heterogeneidade morfológica e sobreposição histológica entre os vários subtipos de tumores. Neste estudo foi utilizada a técnica de hibridação genômica comparativa de alta resolução (HR-CGH) em 64 amostras de TGS, incluindo: 27 adenomas pleomórficos (AP); 11 tumores de Warthin (TW); seis carcinomas ex-adenomas pleomórficos (CXAP); seis carcinomas adenóides císticos (CAC); quatro carcinomas mucoepidermóides (CME); três carcinomas ductais salivares (CDS); três adenocarcinomas (ACAR); dois oncocitomas (ON) e dois carcinomas epiteliaismioepiteliais (CEM). Destas, 47 amostras eram provenientes de tecidos fixados em formalina e em blocos de parafina (FFEP) e 17 de tecidos a fresco. Após a microdissecção, o DNA genômico foi amplificado e marcado por técnica baseada na PCR (SCOMP) e por nick translation, respectivamente. Os cromossomos foram cariotipados usando a imagem DAPI invertido e a intensidade do sinal de hibridação foi determinada ao longo de cada cromossomo. Uma biblioteca com amostras de DNA normais foi construída para selecionar os limites superiores e inferiores para perdas e ganhos cromossômicos. Em todos os casos, as perdas genômicas foram observadas mais freqüentemente do que os ganhos. Na análise comparativa das regiões mínimas comuns detectadas pela HR-CGH envolvendo todos os tipos tumorais, foram observadas alterações genômicas comuns aos diversos tipos, como também exclusivas para os diferentes tipos histológicos. As regiões genômicas consistentemente alteradas nos dois subgrupos tumorais com maior número amostral (adenomas pleomórficos e tumores de Warthin) foram investigadas em bancos de dados para a seleção de genes candidatos que pudessem estar relacionados com a etiologia destes tumores. Os genes NEDD9 (6p24-p25), PPARG (3p25) e c-MYC (8q24.21) foram... / Salivary gland tumors (SGT) are uncommon lesions showing morphologic heterogeneity and histologic overlap among various tumoral subtypes. In the present study, high resolutioncomparative genomic hybridization (HR-CGH) method was performed in 64 SGT samples, including 27 pleomorphic adenomas (PA), 11 Warthin’s tumors (WT), six carcinoma ex pleomorphic adenoma (CXPA), six cystic adenocarcinomas (CAC), four mucoepidermoid carcinomas (MEC), three salivary ductal carcinomas (SDC), three adenocarcinomas (ACAR), two oncocitomas (ON), and two epithelium-myoepithelium carcinomas (EMC). Among these samples, 47 were formalin-fixed and paraffin embedded tissues and 17 were fresh tissues. After laser capture microdissection (LCM), the DNA samples were amplified and labeled by PCR-based methods (SCOMP) and nick translation reaction, respectively. Chromosomes were karyotyped based on their inverted DAPI image and the relative hybridization signal intensity was determined along each chromosome. A library with differentially labeled normal samples was built to select the superior and inferior limits for chromosomal gains and losses. Genomic losses were more frequently observed than gains. The comparison among all tumor samples showed common chromosomal imbalances and also exclusive genomic alterations related to the different histological tumor types. Non-random genomic regions involved in pleomorphic adenomas and Warthin´s tumors were investigated in databases in order to select candidate genes that could be related to the etiology of these tumors. The NEDD9 (6p24-p25), PPARG (3p25) and c-MYC (8q24.21) genes were selected and the gene transcript levels were evaluated by quantitative real time PCR (qRT-PCR) in PA samples. In accordance to the HR-CGH results, it was observed high transcript levels of NEDD9 in 17 out of 37 PA; low levels of PPARG was detected in 11 out of 36 samples; and high levels... (Complete abstract click electronic access below)

Identifying the molecular mechanisms behind reproductive isolation between closely related yeast species provides avaluable understanding of their evolution. Sequence divergence and chromosomal rearrangements are the main post-zygotic barriers behind reproductive isolation within Saccharomyces „sensu strico’ species, where hybrids are readily formed but sterile upon meiosis. Saccharomyces paradoxus and Saccharomyces cariocanus have an almost identical genome in terms of sequence, and therefore provide good model systems to explore the impact of karyotypic rearrangements on reproductive isolation. According to the biological species concept they are considered two different species despite having low sequence divergence. Since the karyotypic analysis revealed that the genomic differences are restricted to four chromosomal translocations, we hypothesized that such rearrangements may be the cause of low spore viability between them. To test this expectation, we engineered two chromosomal translocations in S. paradoxus YPS138, via Cre-loxP mediated recombination event, to render those parts of genome collinear to S.cariocanus UFRJ50816. Our analysis revealed that hybrids between S. cariocanus and engineered S. paradoxus harbouring two translocations showed a significant increase in spore viability (12.7%) compared to control hybrids harbouring five translocations (3.4%) (P=0.0031and P=0.0125, respectively, Two-sample t-test). Consequently, fitness in meiosis was improved four fold by undoing two translocations. Given this result, the prediction for spore viability in complete collinear crossing would be around 50.8 %, which is still far from the value of ca. 100%, which would be expected for strains with very low sequence divergence and belonging to the same species. This indicates that other factors may contribute to meiotic fitness in these hybrids. Further investigation was carried to determine the genome structures by using the PacBio sequencing approach. Our DNA sequencing data revealed other, previously undetected, rearrangements in S. cariocanus strain: one new reciprocal translocation between chromosomes XIII and XIV and 11 inversions distributed in 6 chromosomes. The variations in meiotic viability observed in the engineered hybrids could be because of these 5 chromosomal translocations. Further experiments were also carried out to evaluate the impact of translocations on mitotic fitness and gene expression; we observed a significant drop in the mitotic fitness of engineered translocant strains under different nutritional and temperature stresses. These changes were also accompanied with alteration in genes expression throughout the genome. Our RNA- seq data revealed that many genes were up- or down- regulated because of the translocation. Several genes with altered expression in translocant strains are correlated with morphology changes when they are up- or down- regulated. Therefore, the cell morphology was evaluated under light microscopy and different abnormal cells were detected compared to the wild type. Irregular cell morphology included elongated and clumped cells. Overall, these data confirmed that chromosomal translocations were the cause of reproductive isolation between S. paradoxus and S. cariocanus and play an important role in altering the phenotype and gene expression.