Mouse PEA3 is the founding member of the PEA3 subfamily of ETS transcription factors that includes ERM and ER81. Numerous studies implicate PEA3 subfamily members in a diversity of human cancers, especially breast cancer. Dominant-negative PEA3 (L1NPEA3En) effectively represses activated transcription by all three PEA3 subfamily members. When expressed under control of the MMTV promoter, L1NPEA3En significantly delays the appearance of mammary tumors and reduces their number and size in mouse models of HER2 mediated breast cancer. In addition, L1NPEA3En is not expressed in the mammary tumors that do develop in these mice. These findings strongly suggest a required role for PEA3 subfamily members or other ETS proteins with similar DNA binding specificity in HER2-mediated oncogenesis. The primary objective of this research was to identify the PEA3 subfamily target genes that could play a role in the initiation and progression of tumors, specifically in the breast. To achieve this, a recombinant adenovirus carrying L1NPEA3En was constructed to express L1NPEA3En in three human mammary tumor cell lines: MDA-MB-468, BT-549 and MDA-MB-361. Gene expression analysis using Affymetrix® GeneChip® technology identified a common set of 39 downregulated and 2 upregulated genes in cells expressing L1NPEA3En compared to control cells in all three tumor cell lines. Differentially expressed genes included some that have been shown to play key roles in tumorigenesis such as activating transcriptionfactor 3, heat shock 70kD protein lA and interleukin-8. In addition one colon carcinoma cell line, SW620, was used for gene expression analysis and 7 genes identified in the mammary tumor cell lines were also identified in the colon carcinoma cell line. The results suggest a role for PEA3 subfamily genes in a multiple human cancers mediated through a small subset of common target genes. The genes identified as being differentially expressed by ~NPEA3En hold potential value not only as targets for therapeautic drug discovery, but also as diagnostic or prognostic markers for human cancers, specifically breast cancer. / Thesis / Master of Biological Science (MBioSci)

Previous studies in this laboratory have identified two phosphate transport systems in Sinorhizobium meliloti encoded by the phoCDET and orfA-pit genes respectively. The PhoB regulatory protein is required for transcriptional activation of the phoCDET genes but repress the transcription of orfA-pit. Determination of the DNA sequence upstream of phoU-phoB revealed the presence of genes homologous to the pstA-pstB genes, which encode components of an ABC-type high affinity Pi specific transport system in E. coli. Further analysis of sequence from the S. meliloti genome project (unpublished) revealed the phoR-pstS-pstC genes upstream of pstA-pstB. Using an R-prime approach, we cloned a 7.5 kb Hindlll gene fragment which included the above phoR-pstS-pstC-pstA-pstB and partial phoU genes. Using Tn5-B20 and lacz-aacc1 cassette gene disruption/fusions, we mutated pstA, pstB and phoR gene respectively. We found that: a) pstA-pstB-phoU-phoB are in one operon, b) pstB expression is not regulated by the media phosphate concentration and is independent of phoB, c) in free-living cells, pstB mutants, like phoU or phoB mutants, exhibit alkaline phosphatase negative phenotypes, d) in plant tests, a pstB mutant had normal nitrogen fixation ability and like phoB mutations, the pstB mutation suppressed the Fix- phenotype of phoCDET mutants, e) phoB expression is neither regulated by phosphate concentration nor does its expression appear to be auto-regulated, and f) a phoR mutant exhibited an alkaline phosphatase negative phenotype. Sequence analysis showed that there is no pho box in the upstream of pstA-pstB-phoU-phoB operon and the phoR, but pstS gene has one putative pho box in its promoter region. Also discussion and some ideas for future study were presented. / Thesis / Master of Science (MS)

This thesis is missing page 129. Other copies of this thesis do not have the page either. -Digitization Centre / E. coli expresses an alternative sigma factor, RpoS, in response to starvation and environmental stresses. RpoS is a global regulator and it controls numerous genes, which aids in counteracting these stresses. The RpoS regulon is large but is not completely characterized. We have previously identified over one hundred RpoS-dependent fusions in a genetic screen based on the differential expression of an operon-lacZ fusion bank in rpoS mutant and wild type backgrounds. Forty-eight independent gene fusions were identified including several in well-characterized RpoS-regulated genes such as osmY, katE and otsA. Many of the fusions mapped to genes of unknown function or to genes that were not previously known to be under RpoS control. Based on the homology to other known bacterial genes, some of the RpoS regulated genes with unknown functions may be important for nutrient scavenging. To gain a better insight into the functions of these poorly characterized genes, we tested the ability of the fusion mutants to utilize various carbon sources and to utilize individual amino acids as carbon and nitrogen sources. The results indicate that most of the strains in rpos-backgrounds exhibited better growth in succinate and fumarate and in several amino acids than did the corresponding strains in wild-type backgrounds. / Thesis / Master of Science (MS)

El girasol (Helianthus annuus L.) es uno de los cultivos más importantes de Argentina. La superficie sembrada se ha reducido en los últimos diez años, con el advenimiento de los cultivos genéticamente modificados como la soja, más rentables para los productores agrícolas. Desde entonces se han realizado grandes esfuerzos para recuperar el nivel de producción del cultivo de girasol. Una limitante para la introducción de nuevas tecnologías en este cultivo tradicional es la presencia de parientes silvestres sexualmente compatibles, potenciales receptores de polen, en el área dedicada al cultivo de girasol. Helianthus annuus y H. petiolaris, especies silvestres anuales, diploides y autoincompatibles se han establecido en Argentina en los últimos 60 años. La primera fue introducida intencionalmente como forrajera mientras que la segunda entró probablemente como un contaminante de semillas. Más tarde fueron importadas desde EEUU como fuente de germoplasma para conferir resistencia a patógenos en el mejoramiento de variedades de girasol. Actualmente, ambas especies se encuentran naturalizadas en la región central de Argentina, superponiéndose ampliamente con el área del cultivo. Los objetivos generales de esta tesis fueron obtener información acerca del rango de dispersión de las poblaciones silvestres de girasol en Argentina, estudiar la hibridación y potencial flujo génico entre ellas y el girasol cultivado; en ese caso, estimar la probabilidad de modificación de la aptitud biológica de las poblaciones silvestres que pudiera producir el flujo génico proveniente de las nuevas variedades de girasol. Para ello se realizaron exploraciones y actividades experimentales que permitieran: describir la morfología y distribución de las poblaciones de ambas especies silvestres; comparar la variabilidad genética de marcadores moleculares en poblaciones silvestres procedentes de distintas regiones; identificar caracteres morfológicos intermedios y analizar la fertilidad en plantas presuntamente híbridas; estimar la frecuencia de cruzamientos y tasa de flujo génico desde el girasol domesticado hacia H. annuus silvestre mediante análisis de marcadores moleculares; estudiar la transferencia del carácter resistencia a imidazolinonas desde el cultivo de girasol a H. annuus silvestre y evaluar su efecto sobre la supervivencia y la fecundidad. Se encontraron numerosas poblaciones de Helianthus annuus en las provincias de Córdoba, La Pampa, Buenos Aires, Entre Ríos, Mendoza, San Luis y San Juan. En tanto H. petiolaris fue hallada principalmente en las provincias de La Pampa, San Luis y el oeste de Buenos Aires, sobre suelos arenosos. Ambas especies pueden hibridar con el girasol domesticado y se hallaron plantas con morfología intermedia en 15 departamentos provinciales. Las especies silvestres también son simpátricas en numerosas localidades de la región central del país. La variación genética en 20 poblaciones silvestres y en comparación con el girasol domesticado se investigó mediante marcadores isoenzimáticos y microsatélites (SSR). Los valores de diversidad hallados en este estudio para H. annuus fueron A=1,6, P=0,43 y H=0,17, levemente menores a los informados en su centro de origen y similares a los encontrados en Australia donde H. annuus silvestre fue introducido con fines ornamentales. La progenie de 33 plantas atípicas coleccionadas en 14 sitios representativos del área de difusión fue estudiada para confirmar su origen híbrido. Supervivencia, germinación, rasgos morfológicos y días a floración confirmaron la hibridación entre el cultivo y ambas especies silvestres cuando las familias fueron comparadas con ocho entradas silvestres. Algunas plantas se identificaron como presuntos híbridos entre H. annuus domesticado y ambas especies silvestres, otras como generaciones avanzadas de un genotipo cultivado. A partir del análisis de progenies se determinaron cuatro clases de híbridos de acuerdo a su grado de fertilidad, demostrando que la hibridación y el flujo génico son fenómenos recurrentes entre estas especies en la región central de Argentina. Con el propósito de cuantificar el flujo génico entre el girasol cultivado y silvestre se sembró un campo experimental de girasol circundado por parcelas de girasol silvestre a distancias crecientes. La tasa de hibridación se estimó utilizando un marcador isoenzimático específico de la variedad cultivada. El promedio de híbridos silvestre-cultivo hallado entre la progenie fue de 7%. Las frecuencias más altas de flujo génico (18%) se encontraron en las parcelas más cercanas al cultivo (3m) y decrecieron gradualmente hasta los 500m. La tecnología Clearfield (resistencia a herbicidas de la familia imidazolinonas) ha sido incorporada en los ultimos años en los híbridos comerciales argentinos. Este rasgo resultó adecuado para estudiar los cambios en la aptitud biológica y el posible impacto ambiental que generaría la transferencia de genes desde el cultivo a sus parientes silvestres. Se utilizaron cinco poblaciones de H. annuus silvestre que comprendían el rango de distribución de la especie. Se registró un promedio de 2% de sobrevivientes en 301 plantas de girasol silvestre evaluadas, mientras que la supervivencia de F1 aumentó a 38% sobre un total de 350 plantas. Las retrocruzas con ambos parentales mostraron valores de resistencia al herbicida superiores a 80%. La aptitud biológica medida en todas las generaciones demostró que si bien los híbridos F1 poseían baja fertilidad (estimada mediante la producción de semilla), en las siguientes generaciones se recuperaba la producción de semillas viables. / Sunflower (Helianthus annuus L.) is one of the main crops in Argentina. In the last ten years a reduction in the cultivated acreage took place as some genetic modified crops like soybean became more profitable for the farmers. Since then many efforts have been made to recover the yield levels of sunflower crop. One caveat to introduce new technologies in this traditional crop is the presence of wild relatives, potentially recipients of crop genes through pollen flow. Helianthus annuus and H. petiolaris, two annual, diploid and self-incompatible wild species were established in Argentina in the last 60 years. The former was intentionally introduced as a forage species while the latter probably entered as a seed contaminant. Later, both species were imported from USA as germplasm sources to confer pathogen resistance to sunflower varieties. At present, both wild species are naturalized and widespread over the central part of Argentina where they extensively overlap the sunflower crop region. The goals of the present study were to obtain information about the dispersion range of wild sunflower populations in Argentina, to study hybridization and potential gene flow between them and the cultivated sunflower, and in such situation, to estimate the likelihood of fitness modification in wild populations due to gene flow from the novel sunflower varieties. Explorations and experimental activities were done in order to describe the morphology and population distribution of both wild species; to compare genetic variability of molecular markers in wild populations from different places; to identify intermediate morphological characters and to study fertility in presumed hybrid plants; to estimate hybridization frequencies and gene flow rates from domesticated sunflower to wild H. annuus through molecular marker analysis; to study the transference of the imidazolinone resistant trait from the crop to wild H. annuus, and to evaluate its effects upon survival and fecundity. Several wild Helianthus annuus populations were found in the provinces of Córdoba, La Pampa, Buenos Aires, Entre Ríos, Mendoza, San Luis and San Juan; while H. petiolaris was mainly found on sandy soils in La Pampa, San Luis and western Buenos Aires. Both species can hybridize with the domesticated sunflower and morphologically intermediate plants were found in 15 counties. These wild species are also sympatric in several localities in the central part of the country. Genetic variation in 20 wild populations as compared with domestic sunflower was assessed through isozyme and SSR markers. H. annuus diversity values were A=1.6, P=0.43, and H=0.17, slightly lower than levels reported for the center of origin and similar to those found in Australia, where wild H. annuus was introduced with ornamental purposes. The progeny of 33 off-type plants collected in 14 representative sites of the diffusion area were studied to confirm their hybrid origin. Survival, germination, morphological traits and days to flowering confirmed hybridization between the crop and both wild species, when families were compared with eight accessions of typical wild plants. Some progenies were presumably cropwild H. annuus and H. petiolaris hybrids, and others were advanced generations of a cultivated genotype. From progeny tests, four classes of hybrids were defined mainly according to fertility levels, showing that hybridization and gene flow are recurrent events among these species in the central part of Argentina. In order to measure gene flow between cultivated and wild sunflower, an experimental stand of sunflower was sown surrounded by plots of wild plants at increasing distances. Hybridization rates were estimated using a crop specific isozyme marker. A mean of 7% progenies were crop-wild hybrids. The highest percentage (18%) of gene flow was found among the nearest wild plants (3 m) from the crop and gradually decreased with distance, up to 500 m. The Clearfield technology (resistance to imidazolinone herbicides) has been incorporated to Argentinean commercial hybrids in the last years. This trait was suitable to study fitness changes and the likely environmental impact produced by gene transfer from the crop to its wild relatives. Five populations of wild H. annuus comprising the species ditribution range were used in this study. A mean of 2% surviving wild sunflower plants among 301 screened was found, whereas survival of F1 plants increased to 38% among 350 screened plants. Backcrosses with both cultivated and wild parents showed more than 80% of IMI-resistant offspring. Fitness in all evaluated generations demonstrated that although F1 progenies had low fertility (assessed through seed set), viable seed production was recovered in the following generations.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Source: Masters Abstracts International, Volume: 46-04, page: 2093. Advisers: Xianglin L. Du; Karen H. Lu. Includes bibliographical references.

O câncer de mama é uma das neoplasias malignas mais comuns que afetam mulheres de todo o mundo. No Brasil, o Estado do Rio Grande do Sul tem índices de incidência e mortalidade por câncer de mama que situam-se entre os maiores do país. Aproximadamente 5-10% dos diagnósticos são causados por mutações germinativas em genes de predisposição entre os quais estão BRCA1 e BRCA2, associados à Síndrome de Câncer de mama e Ovário Hereditários (Hereditary Breast and Ovarian Cancer Syndrome ou HBOC, OMIM #114480).A identificação dos casos hereditários de câncer de mama é importante porque indivíduos afetados apresentam risco cumulativo vital muito superior ao da população para o desenvolvimento de câncer, porque familiares de um afetado podem estar igualmente em risco porque há medidas de rastreamento intensivo e intervenções preventivas que podem diminuir significativamente o risco de câncer em portadores de mutação. O diagnóstico molecular da síndrome HBOC é laborioso e caro devido à heterogeneidade molecular da doença. Famílias que apresentam características indicativas de uma síndrome de predisposição ao câncer de mama e ovário hereditários, mas que são negativas para mutações pontuais em BRCA1/2 vêm sendo testadas para grandes rearranjos visto que essas anormalidades têm sido consideradas como respondendo por, no mínimo, 10% do todos os casos HBOC com mutação identificável, incluindo grandes deleções ou duplicações. Um estudo recente de Portugal, demonstrou que um rearranjo fundador no exon 3 de BRCA2 ocorre em por 8% das famílias HBOC do Norte do país. Os objetivos deste trabalho incluíram a verificação da freqüência e caracterização de rearranjos gênicos nos genes BRCA1 e BRCA2, incluindo a mutação fundadora c.156_157insAlu no exon 3 de BRCA2 em famílias brasileiras dealto risco para a síndrome HBOC. Em um grupo de 145 indivíduos em risco nãorelacionados rastreados para a mutação fundadorac.156_157insAlu no exon 3 de BRCA2 foram encontrados 3 portadores da mutação (prevalência de 2%). Em um grupo de 145 indivíduos de risco não-relacionados rastreados para rearranjos gênicos em BRCA1 e BRCA2 pela técnica de MLPA (multiplex ligation-dependent probe amplification) foram identificados 4 portadores de mutação germinativa, sendo a mutação em dois deles um rearranjo gênico no gene BRCA1 (1,4%) envolvendo sequencias Alu. Rearranjos gênicos em BRCA1 e BRCA2 são responsáveis por uma parcela das mutações em famílias HBOC Brasileiras. O presente estudo, envolvendo uma série grande de famílias com o fenótipo da síndrome HBOC, não identificou novos rearranjos fundadores, no entanto, demonstrou a presença de rearranjos tanto em BRCA1 quanto em BRCA2, reiterando a importância da busca ativa por estas alterações, que dificilmente são identificadas por técnicas convencionais de sequenciamento gênico. A técnica de MLPA associada a um protocolo específico para detecção da mutação fundadora Portuguesa c.156_157insAlu podem ser utilizadas como estratégia inicial de rastreamento de mutações em famílias Brasileiras com a síndrome. Os resultados apresentados aqui, no entanto, indicam que mutações serão identificadas em menos de 10% dos casos utilizando esta estratégia. / Breast cancer is one of the most common malignancies affecting women worldwide. In Brazil, the State of Rio Grande do Sul has incidence rates and mortality from breast cancer are among the largest in the country. Approximately 5-10% of the cases are caused by germline mutations in predisposing genes including BRCA1 and BRCA2 are associated with the syndrome of breast and ovarian cancer Hereditary (Hereditary Breast and Ovarian Cancer Syndrome or HBOC, OMIM # 114480). The identification of inherited cases of breast cancer is important because affected individuals have cumulative risk life much higher than the population for developing cancer because of an affected family may also be at risk because there are measures of intensive screening and preventive interventions that can significantly decrease the risk of cancer in mutation carriers. The molecular diagnosis of HBOC syndrome is laborious and expensive due to the molecular heterogeneity of the disease. Families that have characteristics indicative of a cancer predisposition syndrome of hereditary breast and ovarian cancers, but are negative for mutations in BRCA1/2 have been tested for large rearrangements because these abnormalities have been identified as accounting for at least 10 % of all cases HBOC identifiable mutation, including large deletions or duplications. A recent study from Portugal, the founder showed that a rearrangement in exon 3 of BRCA2 occurs in 8% of HBOC families of the north. The objectives of this work included the verification of the frequency and characterization of gene rearrangements in BRCA1 and BRCA2 genes, including c.156_157insAlu founder mutation in exon 3 of BRCA2 mutations in Brazilian families at high risk for HBOC syndrome. In a group of 145 individuals at risk unrelated traced to c.156_157insAlu founder mutation in exon 3 of 3 found BRCA2 mutation carriers (prevalence 2%). In a group of 145 individuals at risk unrelated screened for gene rearrangements in BRCA1 and BRCA2 by the technique of MLPA (multiplex ligationdependent probe amplification) identified four carriers of germline mutation, and two of the mutation in a gene rearrangement in the gene BRCA1 (1.4%) involving Alu sequences. Gene rearrangements in BRCA1 and BRCA2 account for a portion of HBOC mutations in Brazilian families. This study, involving a large series of families with HBOC syndrome phenotype, no new rearrangements identified founders, however, showed the presence of rearrangements in both BRCA1 and BRCA2, reiterating the importance of active search for these changes, which hardly are identified by conventional techniques of gene sequencing. The technique of MLPA protocol associated with a specific mutation detection founder Portuguese c.156_157insAlu strategy can be used as initial screening for mutations in families with Brazilian syndrome. The results presented here, however, indicate mutations that will be identified in less than 10% of the cases using this strategy.

Submitted by Hiolanda Rêgo (hiolandarego@gmail.com) on 2018-02-05T14:14:00Z No. of bitstreams: 1 Dissertação_ICS_ ROCHA, D. J. P. G..pdf: 4034030 bytes, checksum: 7085be9f2fda81bc8248196d4b4e6988 (MD5) / Made available in DSpace on 2018-02-05T14:14:00Z (GMT). No. of bitstreams: 1 Dissertação_ICS_ ROCHA, D. J. P. G..pdf: 4034030 bytes, checksum: 7085be9f2fda81bc8248196d4b4e6988 (MD5) / CNPQ / O método mais comum de normalização em ensaios de quantificação relativa da expressão gênica por PCR quantitativa em tempo real (qPCR) faz uso de um gene de referência, que deve manter sua expressão estável sob diferentes condições experimentais. Em uma meta-análise da literatura e banco de dados de experimentos de análise de expressão gênica em bactérias, os genes gyrA (DNA gyrase subunidade A), gyrB (DNA gyrase subunidade B), dnaG (DNA primase), era (GTPase era) e secA (translocase proteica subunidade secA), figuram entre os mais estáveis em diversas condições experimentais. Neste cenário, este estudo se propõe a desenvolver e construir um conjunto de primers universais para esses genes de referência a partir de organismos modelo de grupos bacterianos de interesse clínico e biotecnológico. As ferramentas de alinhamento, ClustalOmega e PCR virtual, Primer-Blast foram utilizadas para encontrar regiões conservadas entre os genes candidatos em diferentes organismos bacterianos. Dentro do complexo Mycobacterium tuberculosis, todos os genes apresentaram homologia suficiente para o desenho de primers universais, enquanto que em outros grupos bacterianos a homologia de sequência foi restrita a algumas espécies. Potenciais primers universais foram desenhados para diferentes grupos bacterianos e os primers para a família Enterobacteriaceae foram validados por RT-qPCR em Escherichia coli; os resultados foram comparados contra o gene comumente usado, 16S rRNA. Os primers testados apresentaram eficiências de amplificação dentro dos limites esperados e as expressões dos genes de referência foram estáveis nas condições estudadas, tendo sido o gene dnaG o mais estável, de acordo com os softwares NormFinder e RefFinder. Conclui-se que é possível desenhar primers universais funcionais para normalização de RT-qPCR em grupos bacterianos específicos, contudo o baixo nível de conservação gênica de determinados genes pode limitar suas utilizações.