Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death.

Programmed Cell death (PCD) is a highly conserved and genetically controlled event that plays important roles in animal development, homeostasis and disease. Our first objective was to discover and characterize new genes involved in PCD. Since many PCD genes are conserved in Drosophila, and steroid-induced PCD of larval salivary glands (SGs) is transcriptionally regulated with features of both apoptosis and autophagy, we used this exceptionally well-suited in vivo system and performed Serial Analysis of Gene Expression (SAGE) in three pre-death stages. SAGE identified 1244 expressed transcripts, including genes involved in autophagy, apoptosis, immunity, cytoskeleton remodeling, and proteolysis. Of the 1244 transcripts, 463 transcripts belonged to knownlpredicted genes and were 5-fold differentially expressed prior to cell death. Next, we investigated the role of differentially expressed genes from SAGE, in cell death or cell survival, by RNA interference (RNAi ) in l(2)mbn haemocyte Drosophila cells. l(2)mbn cells undergo morphological changes in response to ecdysone treatment, and ultimately undergo PCD. We used cell viability, cell morphology, and apoptosis assays to identify the death-related genes and determined their ecdysone dependency and function in cell death regulation. Our RNAi screen identified six new pro-death related genes, including SH3PXJ and Soxl4, and 21 new pro-survival genes including SoxN. Identification of Soxl4 as pro-death and SoxN as pro-survival suggests that these Sox box proteins may have opposing roles in ecdysone-mediated cell death. Our final objective was to elucidate the function of CG409], a Drosophila homologue of human TNF-alpha induced proteins 8 (TNFAIP8) we identified from SAGE. We created loss-of-function and overexpression mutants of CG4091 to study gene function in vivo and employed immunoprecipitation and mass-spectrometry assays to identify proteins interacting with CG409] in vitro. We identified two proteins that are involved in n-fatty acid oxidation and several cytoskeletal proteins as interaction partners. Immunofluorescence based assays in vivo and in vitro revealed that CG409] is necessary for cytoskeletal remodeling. Further, defects in CG4091 expression affect cellular functions such as autophagy and lipid metabolism/trafficking that require an intact cytoskeleton. Together, our studies provided new insights into the molecular mechanisms involved in Drosophila SG cell death. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches.

This thesis explores text-music relationships in Dallapiccola’s Goethe Lieder. Though the cycle is based on Goethe’s West-östlicher divan, it was Mann’s novel Joseph und seine Brüder that spurred its inception. This seven-song cycle revolves around Suleika, a character from the biblical love story of Joseph and Potiphar’s Wife. Dallapiccola set this text upon reading Mann’s novel, which stems from the same story; however, Mann portrayed the character of Suleika as a sympathetic lover rather than the traditional evil seductress. By conducting a thorough pitch structure analysis of each song, focusing in particular on motives, symmetry and aggregates, this thesis examines text-music relationships to demonstrate how Mann’s Suleika is musically represented. This thesis illustrates that Dallapiccola’s setting is a musical composite of both Goethe and Mann’s Suleikas and thus sheds new analytical and hermeneutic light on an important work by one of the twentieth-century’s most prominent serial composers.

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches.

Serial Analysis of Gene Expression (SAGE) on Caenorhabditis elegans RNA from FACS sorted embryonic body wall muscle cells has identified nearly 8000 genes expressed in nematode body wall muscle. Approximately 60% of these are genes are expressed at low levels (<5 tags/~50,000-100,000 tag library). Low-abundance transcripts have typically been overlooked since most are considered experimental or contamination errors. Consequently, research has been focused on transcripts that are most enriched in the particular tissue of interest. Here I focus on the analysis of low-expressed transcripts in the muscle SAGE libraries in order to investigate what percentage of these are in fact expressed in muscle and are not false positives. Most well characterized C. elegans body wall muscle genes are not expressed at low levels, therefore I anticipate that focusing on these rarely expressed genes will allow for the identification of muscle components that have been previously unrecognized. RT-PCR was performed on RNA isolated from purified body wall muscle cells to initially estimate what fraction of these low abundance transcripts present in the SAGE data are indeed expressed in muscle. I examined 128 genes, of which 84 were represented by a single SAGE tag. From this initial list, 38% of the low-expressed transcripts were verified for their presence in body wall muscle. Subsequently, reporter GFP fusions were used to deduce if these low-expressed transcripts are indeed expressed in vivo within muscle. Of the low-expressed genes that tested positive via RT-PCR, 42% showed in vivo expression in body wall muscle. When the results from the RT-PCR and in vivo expression experiments are combined, I can extrapolate that at least 16% of low-expressed genes identified by the SAGE libraries are in fact expressed in muscle and are not false positives. RNAi and knockout analysis were performed in order to investigate the role of low-expressed muscle genes in myofilament structure. RNAi results show that 14/34 (41%) of the genes screened had mild defects in myofilament organization. The SAGE libraries identified 6388 low-expressed transcripts, this work suggests that at least 16% (1022 genes) of these are in fact expressed in muscle and may reveal new components previously overlooked by other approaches. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

This thesis explores text-music relationships in Dallapiccola’s Goethe Lieder. Though the cycle is based on Goethe’s West-östlicher divan, it was Mann’s novel Joseph und seine Brüder that spurred its inception. This seven-song cycle revolves around Suleika, a character from the biblical love story of Joseph and Potiphar’s Wife. Dallapiccola set this text upon reading Mann’s novel, which stems from the same story; however, Mann portrayed the character of Suleika as a sympathetic lover rather than the traditional evil seductress. By conducting a thorough pitch structure analysis of each song, focusing in particular on motives, symmetry and aggregates, this thesis examines text-music relationships to demonstrate how Mann’s Suleika is musically represented. This thesis illustrates that Dallapiccola’s setting is a musical composite of both Goethe and Mann’s Suleikas and thus sheds new analytical and hermeneutic light on an important work by one of the twentieth-century’s most prominent serial composers.

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Orientadores: Carmem Silvia Passos Lima, Fernando Ferreira Costa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-09T04:25:25Z (GMT). No. of bitstreams: 1 Ortega_ManoelaMarques_D.pdf: 8882093 bytes, checksum: 7322fec26906786406c5385e9ac32926 (MD5) Previous issue date: 2007 / Resumo: O mieloma múltiplo (MM) é uma doença neoplásica caracterizada pelo acúmulo de plasmócitos na medula óssea (MO) com conseqüente osteólise, comprometimento da hematopoese e da síntese das imunoglobulinas normais e com a produção de imunoglobulina monoclonal ou de seus fragmentos. A sobrevida observada para pacientes com a doença varia de alguns meses a dez anos ou mais, o que impõe a busca de fatores prognósticos para a identificação de grupos que devam receber tratamento mais agressivo para o controle da doença. As anormalidades do cariótipo foram descritas, em geral, como fatores com valor prognóstico desfavorável. Por outro lado, não se encontram suficientemente estabelecidos os valores prognósticos das anormalidades numéricas do cromossomo 17 e das deleções e mutações do gene p53 em pacientes com MM. Frente ao exposto, estes constituíram os objetivos deste estudo. Para cumprir tais objetivos, foram avaliados 60 pacientes com MM no período de março de 1999 a dezembro de 2000. A avaliação das anormalidades numéricas do cromossomo 17 foi realizada por meio da análise citogenética convencional e do método de hibridização in situ com fluorescência (FISH), enquanto que a avaliação das deleções do gene foi realizada por meio do método FISH. As mutações do gene p53 foram investigadas por meio da reação em cadeia da polimerase, do polimorfismo de conformação em hélice simples e de seqüenciamento. Não foram identificadas mutações do gene p53 em qualquer dos pacientes incluídos no estudo. Em contraste, as deleções do gene, predominantemente monoalélicas, foram identificadas em 15,7% deles. Observamos ainda, que os pacientes com a deleção do gene p53 apresentaram menor probabilidade de sobrevida do que aqueles sem a deleção do gene (P= 0,0006). A mediana dos tempos de sobrevida global de pacientes do primeiro grupo foi menor do que a observada em pacientes do segundo grupo (7,5 e 17,0 meses, respectivamente; P= 0,05). Frente a estes resultados, pudemos concluir que a deleção do gene p53 constituiu um fator preditivo de menor sObrevida,em nossos casos / Abstract: Multiple myeloma (MM) is a neoplastic disease characterized by the accumulation of plasma cells in the bone marrow (BM) with consequent osteolysis, comprimising hematopoiesis and the synthesis of normal immunoglobins and the production of monoclonal immunoglobin or its fragments. The survival observed for patients with the disease varies from a few months to ten years or more, which makes the search for prognostic factors for the identification of groups which require more aggressive treatment for the control of the disease. Abnormalities of the karyotype have been described, in general, as factors with desfavorable prognostic value. On the other hand, the prognostic values of the numerical abnormalities of chromosome 17, and of the deletions and mutations of the p53 gene have not been sufficientJy established as prognostic values in patients with MM.Thus, these were the objectives of this study. To view these objectives, 60 patients with MM were evaluated in the period of March, 1999 to December, 2000. The evaluation of the numerical abnormalities of chromossome 17 was performed by cytogenetic analysis and by the fluorescence in situ hybridization method (FISH), whilst the evaluation of p53 deletions was performed by FISH. The evaluation of p53 gene mutations was carried out using polymerase chain reaction, single strand conformation polymorphism and the sequencing. No mutations in the p53 gene were detected in any of the patients enroled in the study. In contrast, deletions of the gene, predominantly monoallelic, were identified in 15.7% of them. Furthermore, we observed that patients with p53 deletions demonstrated a lower probability of survival than those without gene deletion (P=0.0006). The median survival time of the patients of the first group was lower than that observed in the second group of patients (7.5 and 17.0 months, respectively; P= 0.05). From these results, we may condude that deletion of the p53 gene constituted a predictive factor of shorter survival, in our cases / Doutorado / Ciencias Basicas / Doutor em Clínica Médica