The objective of this study was to identify possible roles of the class II antigen-associated, electrophoretically invariant chain (Ii) in class II antigen functions through analysis of the kinetics of synthesis, processing, and turnover of Ii in various cellular systems of Ii hyperexpression. Using [35S]methionine metabolic labeling of microsomal membrane proteins and analysis in one-dimensional SDS polyacrylamide gradient gels and two-dimensional electrophoretic gels, enhanced expression of Ii was demonstrated in leukemic cells of a subset of patients with hairy cell leukemia (HCL), in normal peripheral blood cells freshly transformed with EBV, and in Burkitt's lymphoma cell lines treated with n-butyrate. As part of an initial effort to identify leukemic cell subset-defining, membrane-associated molecules which might then be tested as targets for, or regulators of, the anti-leukemic cell immune response, subsets of HCL patients were identified on the basis of leukemic cell enhanced expression of 35,000 and 15,000 dalton proteins (p35 and p15). A similar enhanced expression of the p35 molecule was demonstrated in peripheral blood or cord blood lymphocytes freshly transformed with Epstein-Barr virus (EBV), as compared to long-term cultured EBV cell lines. Further structural characterization of the HCL p35 by one-dimensional SDS-PAGE and two-dimensional gel electrophoresis of HCL total microsomal membrane proteins, anti-class II antigen immunoprecipitates, and anti-Ii immunoprecipitates showed that the HCL p35 molecule was the human class II antigen-associated Ii chain. In order to determine the functional significance of altered Ii expression on class II antigen function, the relative kinetics of Ii synthesis, processing, and turnover was examined in an in vitro model system of regulated Ii synthesis. Treatment of the Burkitt's lymphoma cell line, Jijoye, and its class II antigen-negative, mutant, daughter cell line, P3HR-1, with 4 mM n-butyrate for 48 hr, enhanced the rate of synthesis of the Ii chain. One-dimensional SDS-PAGE and two-dimensional gel, electrophoretic analysis of anti-Ii and anti-class II antigen immunoprecipitates isolated from pulse-/pulse-chase-/or continuously labeled, control and butyrate-treated P3HR-1 and Jijoye cells demonstrated enhanced levels of synthesis of the dominant, most basic form of Ii in the butyrate-treated samples (to a greater degree in Jijoye cells). The butyrate enhancement of Ii synthesis occurred in the presence or absence of detectable class II alpha and beta chains, as did the relative turnover, although the terminal processing of Ii to an electrophoretically slower and more acidic form was dependent upon its association with class II antigens. The level of the dominant, most basic form of Ii expressed in the hairy leukemic cells equaled that in butyrate-treated Jijoye cells. However, hairy leukemic cell, class II antigen-associated Ii was not terminally processed. The results of these experiments were consistent with the following. In lymphoblastoid cells, two pathways for Ii turnover might exist. One is through association with class II antigen complexes and progressive carbohydrate-chain terminal processing, and a second is not associated with class II antigens and such processing. Class II antigens may direct the processing of Ii towards some function (rather than vice versa). Butyrate treatment rather uniquely enhances the synthesis of Ii in some lymphoid cells in the presence or absence of class II antigens. Hairy leukemic cells demonstrate an inability to terminally process Ii which might be relevant to the function of Ii and/or class II antigens on those cells.

Chemotaxis, the ability of a cell to migrate towards a directional stimulus, is a basic property of virtually all cells at some stage in their development. Chemotaxis is preceded by the development of a polarized cellular morphology. The region of the cell closest to the attractant forms a broad lamellipod. The contents of the cell flow forward into this lamellipod and the rear of the cell becomes constricted into a narrow uropod. These local differences in cell structure and function presumably reflect local differences in cell chemistry, but the chemical processes involved are poorly understood. Ca+2 is known to play a ubiquitous role as an essential second messenger in many cellular processes, but its role in chemotaxis is unclear. While many chemotactic stimuli cause Ca+2 to rise intracellularly, the relationship between this rise in Ca+2 and local changes in cell behavior has been difficult to understand. In my dissertation work, I directly tested the role of cellular Ca+2 changes in polarization and chemotaxis by simultaneously imaging intracellular Ca+2 and cell morphology. This work was carried out on single eosinophils isolated from the newt, Taricha granulosa, because of their large size (~100 um, when polarized) and rapid responsiveness (~20 um/min) to chemotactic stimuli present in newt serum. An imaging system was developed to simultaneously image cell behavior, and intracellular Ca+2 following microinjection of the Ca+2 sensitive fluorescent probe, Fura-2. Cell behavior was quantified from time lapse video images captured by a SIT video camera, stored on a video optical disk recorder, and later digitized for analysis. Quantitation was accomplished by interactively tracing the cell's outline and determining the position of the geometric centroid. Variation in the radius of the outline from the centroid was used to calculate a "polarization index", which could be monitored over time. Cell speed was calculated from the movement of the centroid over time. Agents which are known to interfere with Ca+2 signalling significantly inhibited both the polarization and the movement of cells in response to 10% newt serum. These treatments included: chelation of extracellular Ca+2 with EGTA, the organic Ca+2 channel antagonist, verapamil, the inorganic Ca+2 channel blocker, cobalt, the Ca+2 ionophore, ionomycin, and caffeine, an agent known to release Ca+2 from internal stores. In contrast, the K+ ionophore, valinomycin, and treatment of cells with dibutryl cAMP had no effect on cell behavior. The development of a polarized, motile morphology following stimulation of newt eosinophils with 10% serum was accompanied by a rise in intracellular Ca+2. In addition, Ca+2 in a polarized, moving cell was non-uniformly distributed and periodic elevations in intracellular Ca+2 were seen during changes in cell behavior. In turning cells, Ca+2 was significantly higher than in cells moving in a straight line and there was a clearly detectable gradient of Ca+2 within the cell. The region closest to the new direction of movement had the lowest Ca+2 and the rear of the cell was significantly higher. This gradient persisted following a turn, even though Ca+2 was much lower overall in cells moving in a straight line. A gradient of Ca+2 along the long axis of the cell might be important for the differential regulation of different regions of the moving cell. Loading cells with the cell-permeant, esterified form of Fura-2 revealed a region of high Ca+2 associated with the microtubule organizing center (MTOC). This region was surrounded by a membrane system labeled by the lipid soluble, membrane potential sensitive dye, DiOC6(3). This region of Ca+2 was depleted by caffeine treatment. These observations, coupled with the effects of caffeine on cell behavior, suggest that a Ca+2 storage site associated with the MTOC may play a role in regulating cell polarization and chemotaxis. The effects of releasing "caged calcium" on cell behavior and [Ca2+]i were examined as a means of directly testing the ability of changes in [Ca2+]i to regulate cell behavior. Although photolysis of the compound inhibited cell polarization and movement, technical problems made it difficult to attribute these effects entirely to the release of Ca2+. The results presented here, particularly the gradients of [Ca2+]i which were observed, suggest that local regulation of the cytoplasmic components involved in cell movement by local differences in [Ca2+]i could, in part, explain the regional specialization seen during this process. This form of regulation will be discussed in detail, as will potential mechanisms to test for its function during cell polarization and chemotaxis.

T and B-lymphocytes play an important role in an adaptive immune response. Communication between these two cells may result in either a humoral immune response or tolerance. Communication between T and B-lymphocytes involves a number of inducible cell surface molecules on both T and B-lymphocytes. It was the aim of this project to gain a greater understanding of the role of CD40 in the dynamic communication that occurs between naïve T-lymphocytes and resting B-lymphocytes during cognate communication. Because in vivo antigen specific T-lymphocytes are at low frequency, it is difficult to examine antigen-specific naïve T-lymphocytes. Thus, an in vitro system employing naïve antigen-specific T cell receptor (TCR) transgenic T cells and small resting B-lymphocytes that did not express CD40 was devised to examine the role of CD40 in cognate communication between naïve T-lymphocytes and resting B-lymphocytes. Upon recognition of antigen on resting B-lymphocytes that expressed CD40, T-lymphocytes proliferated, expressed the activation antigens CD69 and CD25, and remained responsive to subsequent antigen challenge. In the absence of CD40, resting B-lymphocytes did not induce sustained proliferation or sustained expression of the activation markers CD69 and CD25 on naïve T-lymphocytes, and their recovery was decreased compared to naïve T-lymphocytes that recognized antigen on resting B-lymphocytes that expressed CD40. Naïve T-lymphocytes, however, remained responsive to subsequent antigen challenge after recognition of antigen on resting CD40-/- B-lymphocytes. Recognition of antigen on resting CD40-/- B-lymphocytes also resulted in increased recovery and antigen responsiveness of T-lymphocytes when compared to controls without antigen, The role of CD40 in sustaining activation of naïve T-lymphocytes may be unique to resting B-lymphocytes, since proliferation of naïve T-lymphocytes in response to dendritic cells that did not express CD40 was similar to proliferation of naïve T-lymphocytes in response to dendritic cells that expressed CD40. The mechanism by which CD40 sustained activation of naïve T-lymphocytes was investigated by examining the induction of various costimulatory molecules on resting CD40+/- and CD40-/- B-lymphocytes during cognate interaction with naive T-lymphocytes. Induction of B7-1, upregulation of CD44 and ICAM-1, and sustained but not initial induction of B7-2 required that CD40 be expressed on resting B-lymphocytes. Expression of B7-1 and CD44H was not required for proliferation of naïve T-lymphocytes in response to antigen presented on resting B-lymphocytes. However, sustained expression of B7-2 was crucial for proliferation of naïve T-lymphocytes in response to antigen presented on resting B-lymphocytes.

The encompassing aim of this project was to gain a better understanding of the role of the cellular immune response to dengue virus (DV) infection. Dengue virus occurs as four distinct serotypes, called D1-D4. Symptomatic DV infection occurs as two forms of illness. The more severe form of DV infection, dengue hemorrhagic fever (DHF), is characterized by increased capillary permeability resulting in decreased plasma volume, which may be accompanied by hemorrhagic manifestations. At its most severe, DHF can result in circulatory shock and death. Epidemiological studies indicate that DHF is more likely to occur following a secondary infection with a serotype of DV other than that which caused the primary infection, and there is evidence of increased T cell activation in more severe disease. These data and others indicate that DHF may be of an immunopathological nature. The memory CD4+ T cell response of a D4-immune donor was analyzed. Bulk culture proliferative responses of peripheral blood mononuclear cells (PBMC) to noninfectious DV antigens showed the highest proliferation to D4V antigen, with lesser, crossreactive proliferation to D2V antigen. CD4+ cytotoxic T lymphocyte (CTL) clones were established by stimulation with D4 antigen using a limiting dilution method. Seven out of 15 clones recognized the D4V capsid protein. The clones showed heterogeneity in their usage of T cell receptor Vα and Vβ genes. Six of these CTL clones were crossreactive between 02 and 04, and one clone was specific for D4. Using synthetic peptides, the D4V-specific clone was found to recognize an epitope between amino acids (aa) 47-55 of the capsid protein, while the crossreactive CTL clones each recognized epitopes in a separate location, between aa 83 and 92, which is conserved between D2 and D4. These results showed that the DV capsid protein can be a target of the cellular immune response following DV infection. The bulk culture response of the donor's PBMC to the epitope peptide spanning aa 84-92 was also examined. Peptides containing this epitope induced proliferation of the donor's PBMC in bulk culture, but peptides not containing the entire epitope did not induce proliferation. Also, PBMC stimulated in bulk culture with noninfectious D4V antigen lysed autologous target cells pulsed with peptides containing aa 84-92. These results indicate that this donor exhibits memory CD4+ T cell responses directed against the DV capsid protein and suggest that the response to the capsid protein is dominant not only in vitro at the clonal level, but in bulk culture responses as well. Experiments were performed demonstrating that the CD4+ CTL clones were capable of mediating bystander lysis of non-antigen presenting target cells. Following activation on plate-bound anti-CD3 antibody or in the presence of unlabeled antigen-presenting target cells, these clones could lyse both Jurkat cells and HepG2 cells as bystander targets. Bystander lysis of neighboring, non-infected cells by activated CD4+ CTL clones might contribute to the pathology of DHF. The mechanisms of lysis employed by the T cell clones against both cognate and bystander target cells were assessed using chemical inhibitors of either the perforin- or Fas/FasL-mediated pathways. Three CD4+ CTL clones were demonstrated to lyse cognate, antigen-presenting target cells by a mechanism that primarily involves perforin, while bystander lysis occurred through Fas/FasL interactions. In contrast, one clone used a Fas/FasL mechanism to lyse both cognate and bystander targets. These experiments indicated that the perforin- and FasL-mediated mechanisms of target cell lysis are not mutually exclusive, in that a single clone can kill target cells using either mechanism. Additionally, the ability of CD4+ CTL clones to lyse target cells by the perforin pathway indicates that, like CD8+ CTL, these clones might play a role in viral clearance and recovery from infection through lysis of virus-infected cells. Cytokine production by the capsid-specific CTL clones was also examined. Six of six clones studied produced high quantities of IFN-γ in response to either D2V antigen or the epitope peptide. IFN-γ was also produced by PBMC in a bulk culture from this donor stimulated with D4V antigen. All of the clones produced both TNF-α and TNF-β following stimulation. Four of six clones produced low amounts of IL-2, and only three of six clones produced detectable amounts of IL-4. Production of cytokines by activated CD4+ T cell clones in vivo could contribute to both viral clearance and immunopathology. To better understand the role that cytokine production might play in vivo in response to DV infection, cytokine mRNA levels were examined by PCR in DV-infected Thai children. mRNA for the cytokines IFN-γ, TNF-β, TNF-α, IL-1β, and IL-6 were detectable in the PBMC of DV-infected children. Semi-quantitative PCR analysis indicated that TNF-α mRNA levels were elevated in Thai children with DHF compared to children with classical dengue fever, the less severe form of illness (p=.013). All other cytokines showed no statistically significant difference between children with DHF and those with DF, although IFN-γ showed a trend toward elevation in more severe disease (p=.l). Increased production of TNF-α and/or IFN-γ in vivo could potentially contribute to the immunopathology of severe dengue illness. Taken as a whole, the data presented in this thesis provide a better understanding of the role of the cellular immune response to dengue virus infection and its potential contribution to the immunopathology of dengue hemorrhagic fever.

The temperature-sensitive α-factor receptor (Ste2-3p) and arginine permease (Can1tsp) were found to provide the model substrates for quality control of plasma membrane proteins in Saccharomyces cerevisiae. When the ste2-3 mutant cells were grown at 34°C, Ste2-3p failed to accumulate at the plasma membrane and was delivered to the vacuole for degradation without traversing the plasma membrane. Upon reaching the vacuole, cytoplasmic domains of both Ste2p and Ste2-3p appeared within the vacuolar lumen. Four stp mutants were identified to suppress temperature-sensitive defects in both Ste2-3p and Can1tsp. The stp22 and STP26 mutations also caused missorting of vacuolar protein carboxypeptidase Y, and a subset of vacuolar protein sorting mutants (vps) suppressed ste2-3 mutation. In the stp22 mutant, both Ste2p and Ste2-3p accumulated in the prevacuolar compartment (PVC) and on the plasma membrane. Three independent mutations that bypassed the phenotype of stp22Δ mutant were identified and mapped to the SNF8 locus, and they were found to affect a single amino acid residue (G209D). The mutant protein, Snf8bpp, but not Snf8p, was able to compensate for the lack of functional Stp22p and to restore PVC-to-vacuole trafficking. The order of function for some VPS genes involved in PVC-to-vacuole traffic (class E) was determined by using this special snf8bp allele. In addition, a PtdIns 4-kinase encoded by the PIK1 gene was found to be involved in Ste2-3p trafficking, possibly affecting the PVC function.

The experimental data included in this thesis examines two events involved in signal transduction by the Epidermal Growth Factor Receptor. The first event (receptor oligomerization) occurs at the cell membrane and is proposed to be involved in activating the tyrosine protein kinase activity of the EGF receptor. Activation of the tyrosine protein kinase is an initial step in signal transduction by the EGF receptor. The second event examined (activation of an EGF stimulated serine/threonine protein kinase activity) occurs in the cytosol and may potentially be involved in final transmission of the EGF signal to the cell nucleus. The role of oligomerization in regulating the EGF receptor tyrosine protein kinase was examined by testing two hypotheses: 1) that PMA controls EGF receptor function by regulating the oligomeric state of the receptor and 2) that oligomerization is required to activate the EGF receptor tyrosine protein kinase. The oligomeric state of the EGF receptor was examined by chemical cross-linking and sucrose density gradient centrifugation analysis. It was determined that protein kinase C inhibition of the EGF receptor tyrosine protein kinase activity is independent of the oligomeric state of the receptor. It was also determined that the tyrosine protein kinase of the EGF receptor can be activated in the absence of receptor oligomerization. Threonine 669 is the major site of phosphorylation of the EGF receptor after treatment of cells with EGF. Phosphorylation of this site is also associated with the transmodulation of the EGF receptor caused by platelet-derived growth factor and phorbol ester. The kinetics of activation of this T669 kinase activity is rapid. Furthermore, it was demonstrated that EGF could activate the T669 kinase in the absence of detectable tyrosine kinase activity. Together, these data suggest that the T669 kinase has a role in intracellular signal transduction. Therefore, the T669 kinase was purified and characterized in order to help understand how EGF binding to its receptor at the cell membrane ultimately leads to signal transduction to the cell nucleus.

Chapter II Abstract Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Saccharomyces cerevisiae mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide-induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub1Δ mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show that XPG recruits PC4 to a bubble-containing DNA substrate with a resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate. Chapter III Abstract Previously I established that (1) PC4 significantly suppresses oxidative mutagenesis via its single-strand DNA binding activity, (2) a partial suppression of H2O2-induced lethality was observed in a sub1Δ rad2Δ yeast double mutant compared to the sub1Δ mutant, and (3) PC4 interacts with XPG physically and functionally. These results led me to believe that suppression of oxidative mutagenesis and lethality by PC4 is partially due to its function in an XPG/Rad2-dependent pathway and through additional unidentified mechanism(s). In this chapter, I present studies aimed at investigating different DNA repair pathways in which PC4/Sub1 might participate. I address the possible roles of PC4/Sub1 in transcription-coupled repair (TCR) in terms of its binding specificity to oxidative DNA lesions and its ability to allow efficient resumption of transcription after oxidative DNA damaging treatment. To ask if PC4/Sub1 interacts with other DNA repair proteins to protect cells from oxidative DNA damage, I analyzed spontaneous mutation rates among a series of isogenic, haploid yeast mutant strains deficient of SUB1, base excision repair (BER) and/or nucleotide excision repair (NER) functions. I further analyzed genetic interactions between SUB1 and genes critical to various DNA damage avoidance/tolerance mechanisms, such as mismatch repair (MMR), homologous recombination (HR) and translesion synthesis (TLS).

The aim of this study has been to explore the site specific phosphorylation pattern of rat heart glycogen synthase paying particular attention to phosphorylations that are important to the in vivo control of enzyme activity. This problem has been approached using techniques of immuneprecipitation of 32P labeled synthase from hormonally responsive, freshly isolated adult rat cardiomyocytes. Identification of the active subunit of rat heart glycogen synthase was accomplished by immuneprecipitating synthase from 32P-labeled cardiomyocytes and performing Western blot analysis on DEAE-cellulose fractions containing synthase activity. Using these methods, glycogen synthase activity has been localized to a protein of 88,000 daltons. Reverse phase HPLC analysis of synthase tryptic peptides from either hormone responsive cardiomyocytes or synthase treated in vitro with cAMP-dependent protein kinase and protein phosphatase-1 (PP-1) resulted in finding six reproducible peaks of phosphopeptides. The incorporation of radioactivity into peaks 1 and 2 was associated with both the treatment of cardiomyocytes with epinephrine and the in vitro phosphorylation of rat heart synthase with cAMP-dependent protein kinase. These same two peaks are selectively dephosphorylated when cAMP-dependent kinase treated synthase is incubated with protein phosphatase-1. This dephosphorylation of peaks 1 and 2 are coincident with the conversion of synthase from the D to the I form. Peak 3 is dephosphorylated upon treatment of cardiomyocytes with insulin and hyperphosphorylated in cardiomyocytes derived from alloxan diabetic animals. Taken together these results demonstrates the direct relationship between the phosphopeptides in peaks 1 and 2 and the inhibition of synthase activity in response to epinephrine treatment in the cell. This inhibition can be explained by the activity of cAMP-dependent protein kinase which can duplicate the intracellular, epinephrine-stimulated synthase phosphopeptide pattern. This inhibition can be relieved in vitro by protein phosphatase-1 which dephosphorylates peaks 1 and 2. The effect of insulin and alloxan diabetes is localized to peak 3 whose phosphorylation is unaffected in vitro by either cAMP-dependent protein kinase or protein phosphatase-1.

The mechanism for centrosome assembly in somatic cells has previously been proposed to be microtubule independent. Studies presented in this dissertation demonstrate that in somatic cells pericentrin and γ tubulin, two paradigm centrosome proteins, assemble onto the centrosome in a microtubule, and dynein/dynactin dependent manner. High resolution, three-dimensional, time-lapse digital imaging of pericentrin-GFP labeled centrosomes has revealed tiny particles that move vectorally towards the centrosome at rates exceeding 1μm/second. These pericentrin-GFP particles contain γ tubulin and are not readily visible by standard two-dimensional digital imaging microscopy. Further studies have shown that dynein colocalizes with tiny particles of endogenous pericentrin outside of the centrosome which may reflect assembly intermediates in transit towards the centrosome. Furthermore, when dynein function is disrupted in G1 cells by nocodazole treatment, dynamitin overexpression, or dynein IC antibody (70.1) injection, assembly of pericentrin and γ tubulin onto the centrosome throughout the cell cycle is greatly reduced. Moreover, microtubule co-sedimentation studies have demonstrated that pericentrin associates with microtubules in vitro and is dependent on functional dynein/dynactin. Together these data strongly suggest that pericentrin and γ tubulin are novel cargoes of the dynein/dynactin motor complex which transports these proteins -and likely other components of the 3MDa nucleating complex (Dictenberg et al., 1998)- to the centrosome via rnicrotubules.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A evidencialidade à uma categoria lingÃÃstica utilizada como estratÃgia que permite a manipulaÃÃo das informaÃÃes quanto à fonte do conhecimento e ao grau de (des)comprometimento do produtor com essa fonte. No intuito de investigar a manifestaÃÃo dessa categoria na construÃÃo das dissertaÃÃes acadÃmicas no portuguÃs brasileiro contemporÃneo, objetiva-se: a) rediscutir o estatuto da evidencialidade como categoria lingÃÃstica; b)analisar as relaÃÃes entre modalidade e evidencialidade; c) verificar as estratÃgias utilizadas no uso das marcas de evidencialidade no trabalho cientÃfico de grau, do tipo dissertaÃÃo; d) identificar, descrever e analisar as marcas lingÃÃsticas evidenciais no trabalho cientÃfico de grau do tipo dissertaÃÃo; e) relacionar o uso de evidenciais com estratÃgias para efeito de (des)comprometimento na construÃÃo textual. Ao identificar e interpretar as marcas evidenciais na construÃÃo dos textos acadÃmicos, a pesquisa busca contribuir para a explicitaÃÃo dos efeitos de sentido vinculados à veiculaÃÃo das informaÃÃes de forma estratÃgica, jà que essas marcas sÃo utilizadas com propÃsitos diversificados, como por exemplo: recurso ao chamado âargumento de autoridadeâ, atenuaÃÃo da responsabilidade em relaÃÃo ao que à dito, modalizaÃÃo no contÃnuo entre a certeza e a nÃo-certeza, sinalizando que algo nÃo està sendo dito de forma categÃrica e sugerindo um grau de (des)comprometimento em relaÃÃo à verdade da proposiÃÃo, como tambÃm um posicionamento crÃtico em relaÃÃo à fonte da informaÃÃo, permitindo uma correta avaliaÃÃo do conteÃdo assimilado pelo leitor, dentre outros. A anÃlise, orientada por pressupostos funcionalistas, conta com uma dimensÃo teÃrica, voltada para a rediscussÃo dos limites conceituais entre as categorias modalidade e evidencialidade, e uma dimensÃo analÃtica, que, em constante diÃlogo com a teoria, investiga, qualitativa e quantitativamente, o uso de marcas da evidencialidade na dissertaÃÃo acadÃmica, trabalho que à requisito para a obtenÃÃo do tÃtulo de mestre. A escolha do gÃnero para a constituiÃÃo do corpus justifica-se pela suposiÃÃo de que esse à um dos gÃneros textuais que apresenta maior quantidade de informaÃÃo cuja fonte nÃo à o prÃprio autor, o que condiciona o uso das marcas de evidencialidade na relaÃÃo observÃvel com o grau de (des)comprometimento do produtor relativamente à informaÃÃo veiculada. A obtenÃÃo das 290 ocorrÃncias para esta pesquisa concretizou-se a partir da organizaÃÃo de um corpus constituÃdo por dez dissertaÃÃes acadÃmicas da Ãrea de CiÃncias Humanas, das quais se utilizaram, como material de anÃlise, a introduÃÃo e a conclusÃo. Os resultados obtidos verificaram os efeitos de sentido associados ao uso de meios de expressÃo evidencial na construÃÃo da argumentaÃÃo em dissertaÃÃes, comprovando que a evidencialidade no discurso acadÃmico, em particular no corpus coletado, à mais utilizada para promover um baixo comprometimento, tendo o verbo como o meio lingÃÃstico mais utilizado e as noÃÃes evidenciais inferencial e citativa como as mais freqÃentes. / Evidentiality is a linguistic category used as a strategy to deal with information, considering the producerâs source of knowledge and his extent of (un)commitment to this source. In order to investigate the manifestation of this category in the construction of academic dissertations written in contemporary Brazilian Portuguese, this work aims to: a) rediscuss the status of the evidentiality as a linguistic category; b) analyse the relationship between modality and evidentiality; c) verify the strategies used to indicate evdentiality in scientific works such as dissertations; d) identify, describe and analyse linguistic marks of evidentiality in such works; e) relate the use of evidentials to strategies of (un)commitment in the production of a text. Though the identification of evidentiality marks in academic texts, this work aims to contribute to explicit the sense effects related to the strategic transmition of information, since these marks are used with different purposes, like: use of the authority argument, reduction of responsibility in relation to what is being said; certainty/uncertainty modalization, suggesting that something is being said in a non-categorical way, suggesting a certain degree of (un)commitment in relation to the truth of the proposition, as well as showing a critical position in relation to the source of information, allowing a correct evaluation of the content taken in by the reader; among other purposes. The analysis, based on functionalist presuppositions, rediscusses the conceptual limits between modality and evidentiality. It investigates, in terms of both quality and quantity, the use of evidentiality marks in academic dissertations. This genre was chosen due to the supposition that it is one of the textual genres whose source of information is mostly not the author himself. This fact influences the use of evidentiality marks in relation to the degree of the producerâs (un)commitment to the information provided. The 290 ocurrencies were collected from a corpus which comprises 10 academic dissertations in Humanity Studies. Only the introduction and the conclusion sections of these Works were considered. The results showed that the sense effects are associated to the use of evidentiality expressions in the kind of argumentation process in dissertations. This proves that the evidentiality in the academic discourse, particularly in the corpus collected, is used especially to assure a lower degree of commitment, being the verb the linguistic means most chosen to express this, and the inferential and quotative evidentiality notions as the most frequent ones.