A cell integrates mitogenic signals received at the plasma membrane with intracellular biochemical changes to direct the events of cell division. Oocytes from Xenopus laevis offer a system that allows molecular dissection of pathways controlling cell growth and division in response to extracellular cues. Xenopus oocytes, physiologically arrested in a G2 like state, respond to the hormone progesterone to reinitiate meiosis and mature into a fertilizable egg. Signals received at the oocyte membrane induce translation of dormant maternal mRNAs that not only drive meiotic entry but also maintain the cell cycle arrest in an egg. A major pathway controlling the translation of these mRNAs is cytoplasmic polyadenylation, facilitated by the Cytoplasmic Polyadenylation Element Binding protein (CPEB) through cis-acting elements in their 3'untranslated regions (3'UTRs). Cytoplasmic polyadenylation requires the phosphorylation of serine174 on CPEB by Aurora-A as well as the translation of a hitherto unknown mRNA. The transcript of the RINGO/Spy gene is a putative candidate for this unknown upstream regulator of CPEB function. RINGO/Spy mRNA is translationally repressed in immature oocytes by a ribonucleoprotein (RNP) complex consisting of the repressor Pumilio-2, the putative activator Deleted in Azoospermia-like (DAZl) and embryonic poly A binding protein (ePAB). Progesterone signaling leads to the dissociation of Pumilio-2 from the mRNP and the ensuing RINGO/Spy protein synthesis, in turn, promotes cytoplasmic polyadenylation and oocyte maturation. Pumilio and its associated proteins, such as Drosophila Brain tumor (Brat) and DAZl, in addition to their cytoplasmic roles have ill-defined functions within the nucleus. We detected DAZl within the nucleoli of telomerase-immortalized human retinal pigment epithelial (RPE) cells in interphase and on acrocentric chromosomes during mitosis. DAZl colocalizes with the RNA polymerase I associated Upstream Binding transcription Factor (UBF), most likely through pre-ribosomal RNA and is a likely component of the Nucleolar Organization Region (NOR). Stably knocking down DAZl in RPEs using short hairpin RNAs results in loss of nucleolar segregation, the physiological outcome of which is under investigation. These preliminary findings indicate an additional role for DAZl within the nucleolus, one likely to be independent from cytoplasmic translational control.

The focus of this dissertation is the role of specific CD8+ T cells in the pathogenesis of a highly lethal human viral disease, hantavirus pulmonary syndrome (HPS). HPS is a zoonotic disease caused by transmission of Sin Nombre virus (SNV) from chronically infected deer mice. In humans, this fulminant infection is characterized by lung capillary leakage, respiratory failure and cardiogenic shock. Individuals with HLA-B*3501 have an increased risk of developing severe HPS, and the majority of defined CD8+ T cell epitopes in SNV are presented by this HLA allele, suggesting that CD8+ T cell responses to SNV contribute to pathogenesis. We speculate that CD8+ T cell mediated immune responses to SNV antigens in pulmonary endothelial cells contribute to the pathology of HPS. Specifically, we hypothesize that there are quantitative and/or qualitative differences in SNV-specific CD8+ T cell responses in HPS patients with moderate vs. severe disease. In this dissertation I measured the frequencies of SNV-specific CD8+ T cells during acute HPS. Using HLA/peptide tetramers, I quantitated circulating SNV-specific CD8+ T cells of all the available HLA-B35+ patients with HPS caused by SNV. This is the first time hantavirus-specific T cells have been quantitated during acute infection. I report that between 2.9% and 44.2% of the CD8+ T cells were specific for the three SNV epitopes in combination during acute disease in the patients analyzed in this study. These levels are very high in comparison to the frequencies reported in the literature for other acute human viral infections. Furthermore, I report significantly higher frequencies of SNV-specific T cells in patients with severe HPS requiring mechanical ventilation (up to 44.2% of CD8+ T cells) than in moderately ill HPS patients hospitalized but not requiring mechanical ventilation (up to 9.8% of CD8+ T cells). These results imply that virus-specific CD8+ T cells contribute to HPS disease outcome. In this dissertation I also provide preliminary data on qualitative aspects of SNV-specific T cells. Analysis of the TCR repertoire of SNV-specific T cell lines isolated from the PBMC of acute HPS patients raises the possibility that SNV-specific T cells express a limited number of TCR Vβ alleles; however, this is quite speculative because it is based on the analysis of only seven CTL lines. Analysis of cytokine expression by the CTL lines in response to in vitro antigen-specific stimulation indicate that SNV-specific T cells are capable of secreting IFN-γ, TNF-α, and IL-13 upon stimulation. The data presented in this dissertation extend previous studies, which suggested a role for virus-specific T cells in HPS pathogenesis and support our hypothesis that virus-specific CD8+ T cells contribute to HPS disease outcome. The results of this study will be useful in the design of future therapeutic strategies for this emerging human pathogen. The conclusions of this study may also benefit the study of other human viral hemorrhagic fevers. Improved understanding of the mechanism of pathogenesis of severe viral zoonoses will result in better treatment and prevention strategies.

A potential approach to in vivo gene therapy is to target retrovirus to specific receptors through a ligand-receptor interaction. Previous studies have placed a ligand at or close to the N-terminus of the ecotropic Moloney murine leukemia virus envelope and require co-expression of a wild type envelope on the pseudotyped virus for successful transduction of human cells. In this study, over forty chimeric envelopes were generated, which have single or multiple insertions of a 13 or 21 amino acid RGD containing sequence, flanked by cysteine residues, that target the cellular integrin receptors (Chapter III). Virus displaying only the chimeric envelopes was generated from packaging cell lines that express the gag and pol genes. Many of the mutant envelopes demonstrated the formation of syncytia when they were transfected into the XC indicator cell line, which is frequently used to determine envelope binding and fusion capabilities. Pseudotyped virus for several of the chimeric envelopes, transduced both NIH 3T3 mouse fibroblasts and human A375 melanoma cells. Ligands placed in the N-terminal region, within the VRA variable domain, and close to the N-terminus of the proline-rich region (PRR), demonstrated transduction into human melanoma cells. Ligands placed within the PRR and the C-terminus of the envelope did not demonstrate transduction into melanoma cells, although host cell transduction was demonstrated. Pseudotyped virus expressing an RGE containing target sequence, replacing the RGD sequence, had significantly lower transduction efficiency of melanoma cells. These data indicate that the MLV envelope tropism can be altered by insertion of short ligands at various locations throughout the envelope. These initial results were promising and helped to define regions within the envelope that could accommodate the insertion of small targeting ligands, that could redirect the tropism of pseudo typed virus to human cells. In the second part of this study, the focus shifted to targeting receptors that were expressed on specific cells, such as carcinoma cells. We inserted short ligands, flanked with cysteines, into the envelope to generate numerous targeting constructs that bind to receptors over-expressed on a variety of carcinoma cells. These pseudotyped retroviral vectors were generated by packaging cell lines that express only the viral Gag and Pol genes, with no wild-type envelope present. Select chimeric envelopes that express the 21 amino acid bombesin (BN)/gastrin releasing protein (GRP) binding sequence successfully transduced human melanoma cells, breast cancer cells, and cells that express the cloned GRP receptor gene. Nine additional chimeric envelopes were generated, that express a modified 56 amino acid heregulin sequence (HRG), that targets c-rbB-3 (Her-3) and c-erbB-4 (Her-4) receptors on breast carcinoma cells. Pseudotyped virus expressing only the BN/GRP mutant envelopes, transduced NIH 3T3 host cells, and two human carcinoma cell lines; A375 melanoma and MDA-MB-231 breast cells. The HRG chimeric envelopes demonstrated transduction of NIH 3T3 cells and human MDA-MB-453 breast carcinoma cells. Finally, a pseudotyped virus that expressed the chimeric BN/GRP envelopes and packaged the thymidine kinase gene, transduced melenoma and breast carcinoma cells and demonstrated ganciclovir cytotoxicity. Collectively, these data indicate that ligands of various sizes can be used to target pseudotyped virus to a variety of human cancer cells and transfer genes of interest. These findings may expand the feasibility and potential scope of gene therapy.

Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.

Fully grown oocytes' of the African clawed frog, Xenopus laevis, are arrested at the diplotene stage of meiotic prophase I, which resembles the G2 phase of the mitotic cell cycle. Re-entry into the meiotic divisions is initiated by hormonal signaling normally provided by progesterone. Progesterone signaling leads to the activation of maturation promoting factor (MPF), a heterodimer consisting of the protein kinase cdk1 and cyclin B1; this complex promotes the oocyte's entry into M phase of meiosis I. A crucial event required for MPF activation is cytoplasmic polyadenylation element (CPE)-mediated translation of specific dormant mRNAs such as c-mos and cyclin B1. The CPE, which resides in mRNA 3' untranslated region (UTR), is bound by the CPE binding protein (CPEB), which in turn is bound by Maskin. Maskin is bound to the 5' cap binding protein eIF4E. This type of closed-loop mRNA structure inhibits the recruitment and assembly of the translation initiation complex at the 5'UTR of CPE containing mRNAs. To alleviate this inhibition, CPEB undergoes phosphorylation on S174 by the serine/threonine kinase Aurora A. Phosphorylated CPEB promotes the recruitment of specific polyadenylation factors leading to the polyadenylation of the dormant mRNA, resulting in the disassociation of Maskin from eIF4E. eIF4E is subsequently bound by translation initiation factors leading to mRNA assembly into polysomes and synthesis of the encoded protein. Insulin signaling has also been shown to induce oocyte maturation. However, this signaling cascade uniquely requires the activation of two upstream components, PI3 kinase and PKC zeta. In this thesis, I show that insulin induced oocyte maturation requires the same CPE-mediated mRNA translation mechanism as had been described for progesterone signaling. I also show that Aurora A kinase activation and S174 phosphorylation play an essential role in insulin-induced CPE-mediated mRNA translation. Interestingly, inhibition of PI3 kinase and PKC zeta inhibits CPE-mediated polyadenylation only in the insulin-signaling pathway; the progesterone pathway is unaffected. These results clearly indicate that different upstream signaling components control CPE-mediated translation between progesterone and insulin signaling cascades. However, both pathways are antagonized by over expressed GSK-3, leading to inhibition of oocyte maturation. Furthermore, I found that GSK-3 inhibits Aurora A kinase activity by directly phosphorylating Aurora A on serine 290/291, promoting an inhibitory autophosphorylation event on serine 349. The importance of a GSK-3/Aurora A interaction is underscored by the finding that GSK-3, Axin, and Aurora A reside in a complex in immature oocytes. During progesterone or insulin signaling, GSK-3 dissociates from Aurora A allowing Aurora A to become active, leading to CPEB phosphorylation, CPE-mediated mRNA translation and oocyte maturation.

The development of cancer is a multi-step process which involves a series of events, including activation of oncogenes and loss of tumor suppressor function, leading to cell immortalization and misregulated proliferation. In the last few years, the importance of epigenetic defects in cancer development has become increasingly recognized. While most epigenetic studies focus on silencing of tumor suppressors, this thesis addresses defects in the maintenance of silenced heterochromatin in cancer, particularly breast cancer. Breast cancer is a leading cause of cancer in women and many familial cases have been linked to mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2. BRCA1 has been linked to DNA repair as well as multiple other cellular processes, including cell cycle checkpoints, ubiquitination, centrosome function, and meiotic silencing of the XY body. This work began with a particular interest in the report that BRCA1 was linked to the failed maintenance of random X-inactivation in female somatic cells, via a role in supporting XIST RNA localization to the inactive X chromosome (Xi). XIST RNA is a non-coding RNA that fully coats or “paints” the Xi and induces its silencing. Work presented in Chapter II substantially clarifies the relationship of BRCA1 to XIST RNA, based on several lines of experimentation. Loss of BRCA1 does not lead to loss of XIST RNA in these studies, nor did reconstitution of HCC1937 BRCA1-/- tumor cells with BRCA1 lead to XIST RNA localization on Xi, although an effect on XIST RNA transcription is possible. Studies of BRCA1 localization with Xi showed that BRCA1 has a limited association with the Xi in ~3-10% of cells, it rarely colocalizes with XIST RNA to a significant extent, but rather is in close apposition to a small part of the XIST RNA/Xi territory. Additionally, analysis of several breast cancer cell lines revealed mislocalization of XIST RNA in some breast cancer cell lines. Many studies have examined BRCA1 foci that form following DNA damage and demonstrated that these are sites of repair. However, whether the numerous large foci consistently present in normal S-phase nuclei were storage sites or had any function was unknown. In Chapter III, I demonstrate that the BRCA1 foci in normal S-phase nuclei associate overwhelmingly with specific heterochromatic regions of the genome. More specifically, BRCA1 foci often associate with centromeric or pericentromeric regions in both human and mouse cells. In human cells BRCA1 foci often appear juxtaposed to centromeric signal, whereas in mouse, BRCA1 often rings or paints the large chromocenters, clusters of DAPI-dense pericentric and centric heterochromatin. Using PCNA and BrdU as markers of replication, I demonstrate that BRCA1 preferentially associates with the chromocenters during their replication, although high-resolution analysis indicates that BRCA1 and PCNA foci rarely directly overlap. Interestingly, cells with defects in BRCA1 were found to have lagging chromosomes and DNA bridges which nearly always contained satellite DNA, which is consistent with the possibility that BRCA1 deficit contributes to failed separation of sister chromatids at the centromere. This is consistent with other recent reports that BRCA1 is necessary for DNA decatenation by topoisomerase II during routine replication and with my demonstration that topoisomerase II also accumulates on pericentric heterochromatin (PCH) during replication. Chapter IV presents recent work which reveals that RNA is commonly expressed from the centric/pericentric heterochromatin and appears to be linked to its replication. In mouse cells RNA from heterochromatic sequences is readily detected using a broad molecular cytological assay for repeat transcription (the COT-1 RNA assay). In addition to a more dispersed nucleoplasmic signal from euchromatic nuclear regions, distinct localized foci of repeat RNA are detected with COT1 probe or pancentromeric probe. Further analysis with the minor satellite (centromere proper) and the major satellite (comprising the larger pericentric heterochromatin) reveals that the large RNA foci often contain these satellite sequences, long thought to be essentially silent. These foci generally associate with the PCH of chromocenters, and produce various patterns similar to BRCA1- including a larger signal partially painting or ringing the chromocenter in a fraction of cells. In conjunction again with PCNA staining, it was possible to determine that the major satellite RNAs associate with the chromocenters during replication. While the satellite RNA co-localizes precisely with PCNA, neither of these co-localizes at high resolution with BRCA1, although they all are present on replicating chromocenters contemporaneously. These findings show that satellite RNAs are more widely expressed in normal cells than previously thought and link their expression to replication of centromere-linked heterochromatin. Finally, Chapter V presents three lines of recent results to support a major concept forwarded in this manuscript: that loss of Xi heterochromatin may reflect defects in the broader heterochromatic compartment, which may be manifest at multiple levels. I provide evidence using two new assays that both the peripheral heterochromatic compartment and the expression and silencing of satellite repeats is commonly compromised in cancer, although this appears to vary among cancer lines or types. The final results connect back to the question with which I began: what maintains XIST RNA localization to the chromosome in normal cells. These results demonstrate for the first time that Aurora B Kinase activity, mediated by Protein Phosphatase 1 (PP1) during interphase, controls the interphase retention and mitotic release of XIST RNA from the chromosome, likely linked to chromatin modifications such as H3Ser10 phosphorylation. As Aurora B Kinase is commonly over-expressed in cancer and is linked to chromatin changes, this exemplifies one type of mechanism whereby broad epigenetic changes in cancer may impact XIST RNA localization and the maintenance of heterochromatin more generally. This thesis represents a melding of cancer biology with the study of X inactivation and heterochromatin, with findings of fundamental interest to both of these fields.

The outer dynein arm-docking complex (ODA-DC) specifies the outer dynein arm-binding site on the flagellar axoneme. The ODA-DC of Chlamydomonas contains equimolar amounts of three proteins termed DC1, DC2, and DC3 (Takada et al., 2002). DC1 and DC2 are predicted to be coiled-coil proteins, and are encoded by ODA3 and ODA1, respectively (Koutoulis et al., 1997; Takada et al., 2002). Prior to this work, nothing was known about DC3. To fully understand the function(s) of the ODA-DC, a detailed analysis of each of its component parts is necessary. To that end, this dissertation describes the characterization of the smallest subunit, DC3. In Chapter II, I report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 D protein with four EF hands that is a member of the CTER (Calmodulin, Troponin C, Essential and Regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming due to loss of some but not all, outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly. As mentioned above, DC3 has four EF-hands: two fit the consensus pattern for calcium binding and one contains two cysteine residues within its binding loop. To determine if the consensus EF-hands are functional, I purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of DTT and Mg2+. As reported in Chapter III, the protein bound one calcium ion with an affinity (Kd) of ~1 x 10-5 M. Calcium binding was observed only in the presence of DTT and thus is redox sensitive. DC3 also bound Mg2+ at physiological concentrations, but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium-binding activity of the bacterially expressed protein. To investigate the role of the EF hands in vivo, I transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the E to Q mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains obscure. In Chapter IV, I report the initial characterization of a DC3 insertional mutant having a phenotype intermediate between that of the DC3-deletion strain and wild type. Furthermore, I suggest future experiments that may help elucidate the specific role of DC3 in outer arm assembly and ODA-DC function. Lastly, I speculate that the ODA-DC may play a role in flagellar regeneration.

Many polypeptide growth factors act by binding to cell surface receptors that have intrinsic tyrosine kinase activity. Binding of these growth factors to their cognate receptors results in the initiation of mitogenic signals which then get transduced to the interior of the cell. A critical target for extracellular signals is the nucleus. A plethora of recent evidence indicates that extracellular signals can affect nuclear gene expression by modulating transcription factor activity. In this study, I have determined that the transactivation domain of c-Myc (protein product of the c-myc proto-oncogene) is a direct target of mitogen-activated signaling pathways involving protein kinases. Further, my study demonstrates that transactivation of gene expression by c-Myc is regulated as a function of the cell cycle. c-Myc is a sequence-specific DNA binding protein that forms leucine zipper complexes and can act as a transcription factor. Although, significant progress has been made in understanding the cellular properties of c-Myc, the precise molecular mechanism of c-Myc function in oncogenesis and in normal cell growth is not known. I have focused my attention on the property of c-Myc to function as a sequence-specific transcription factor. In my studies, I have employed a fusion protein strategy, where the transactivation domain of the transcription factor c-Myc is fused to the DNA binding domain and nuclear localization signal of the yeast transcription factor GAL4. This fusion protein was expressed together with a plasmid consisting of specific GAL4 binding sites cloned upstream of a minimal E1b promoter and a reporter gene. The activity of the c-Myc transactivation domain was measured as reporter gene activity in cell extracts. This experimental approach enabled me to directly monitor the activity of the c-Myc transactivation domain. Results listed in Chapter II demonstrate that the transactivation domain of c-Myc at Ser-62 is a target of regulation by mitogen-stimulated signaling pathways. Furthermore, I have determined that a mitogen activated protein kinase, p41mapk, can phosphorylate the c-Myc transactivation domain at Ser-62. Phosphorylation at this site results in a marked increase in transactivation of gene expression. A point mutation at the MAP kinase phosphorylation site (Ser-62) causes a decrease in transactivation. c-Myc expression is altered in many types of cancer cells, strongly implicating c-myc as a critical gene in cell growth control. The molecular mechanisms by which c-Myc regulates cellular proliferation are not understood. For instance, it is not clear where in the cell cycle c-Myc functions and what regulates its activity. In exponentially growing cells, the expression levels of c-Myc remain unchanged as the cells progress through the cell cycle. The function of c-Myc may therefore be regulated by a mechanism involving a post-translational modification, such as phosphorylation. Results described in chapter IV demonstrate that the level of c-Myc mediated transactivation oscillates as cells progress through the cell cycle and was greatly increased during the S to G2/M transition. Furthermore, mutation of the phosphorylation site Ser-62 in the c-Myc transactivation domain diminishes this effect, suggesting a functional role for this phosphorylation site in the cell cycle-specific regulation of c-Myc activity. Taken together, my dissertation study reveals a molecular mechanism for the regulation of nuclear gene expression in response to mitogenic stimuli.

Trata-se de uma pesquisa de caráter quantitativo e qualitativo, composta de um levantamento da pós-graduação brasileira com foco na comparação das áreas Direito, Engenharia Civil e Medicina e por 24 entrevistas semi-estruturadas individuais gravadas com 18 mestrandos e seis orientadores da Faculdade de Direito, da Escola Politécnica e da Faculdade de Medicina da Universidade de São Paulo. O objetivo geral da pesquisa é comparar como se desenvolve o processo de pós-graduação das três profissões mais tradicionais em uma universidade pública de destaque, relacionando o processo de elaboração da dissertação com a concomitante inserção do aluno na atividade profissional. Como resultado destaca-se que os mestrandos seguiriam a carreira acadêmica, mas simultaneamente à carreira profissional. Enquanto os mestrandos de Direito e Engenharia civil acreditavam no mestrado como facilitador para uma melhor inserção no mercado de trabalho, os de Medicina referiam a busca pelo mestrado como conseqüência natural pela sua proximidade com a universidade. Nas três áreas a universidade se defronta com contradições entre a formação de uma elite pensante, a real vocação do curso de pós-graduação stricto sensu na formação de pesquisadores, e sua qualificação profissional segundo um perfil demandado pelo mercado global. / This is a quantitative and qualitative approach research, consisting of a survey of Brazilian postgraduation focused in the comparison of Law, Civil Engineering and Medicine areas and composed by 24 semi-structured recorded individual interviews with 18 Masters students and six advisors of the Faculty of Law, the School of Engineering and the Faculty of Medical Sciences of the University of São Paulo. The general objective of the research was compare the postgraduation developing process of the three most traditional professions in a distinct public university, relating the dissertation elaboration process with the concomitant insertion of the student in the professional activity. As result, was emphasized that Masters students would follow the academic career, but simultaneously to the professional career. While Law and civil Engineering Masters students considered Master Course as facilitative for a better insertion in the job market, Medicine ones related the seek for Master Course as natural consequence by their proximity to the university. In the three areas the university confronts with contradictions between the elite think tank formation, the real vocation of the postgraduation stricto sensu course in the researchers education, and their professional qualification according to a profile demanded by the global market.

Trata-se de uma pesquisa de caráter quantitativo e qualitativo, composta de um levantamento da pós-graduação brasileira com foco na comparação das áreas Direito, Engenharia Civil e Medicina e por 24 entrevistas semi-estruturadas individuais gravadas com 18 mestrandos e seis orientadores da Faculdade de Direito, da Escola Politécnica e da Faculdade de Medicina da Universidade de São Paulo. O objetivo geral da pesquisa é comparar como se desenvolve o processo de pós-graduação das três profissões mais tradicionais em uma universidade pública de destaque, relacionando o processo de elaboração da dissertação com a concomitante inserção do aluno na atividade profissional. Como resultado destaca-se que os mestrandos seguiriam a carreira acadêmica, mas simultaneamente à carreira profissional. Enquanto os mestrandos de Direito e Engenharia civil acreditavam no mestrado como facilitador para uma melhor inserção no mercado de trabalho, os de Medicina referiam a busca pelo mestrado como conseqüência natural pela sua proximidade com a universidade. Nas três áreas a universidade se defronta com contradições entre a formação de uma elite pensante, a real vocação do curso de pós-graduação stricto sensu na formação de pesquisadores, e sua qualificação profissional segundo um perfil demandado pelo mercado global. / This is a quantitative and qualitative approach research, consisting of a survey of Brazilian postgraduation focused in the comparison of Law, Civil Engineering and Medicine areas and composed by 24 semi-structured recorded individual interviews with 18 Masters students and six advisors of the Faculty of Law, the School of Engineering and the Faculty of Medical Sciences of the University of São Paulo. The general objective of the research was compare the postgraduation developing process of the three most traditional professions in a distinct public university, relating the dissertation elaboration process with the concomitant insertion of the student in the professional activity. As result, was emphasized that Masters students would follow the academic career, but simultaneously to the professional career. While Law and civil Engineering Masters students considered Master Course as facilitative for a better insertion in the job market, Medicine ones related the seek for Master Course as natural consequence by their proximity to the university. In the three areas the university confronts with contradictions between the elite think tank formation, the real vocation of the postgraduation stricto sensu course in the researchers education, and their professional qualification according to a profile demanded by the global market.