HIV gp120 binds CD4+ cells within plasma membrane lipid rafts inducing a conformational change in gp120 that exposes its V3 loop that binds to a chemokine co-receptor, also within lipid rafts, and initiates fusion. Glycosphingolipids (GSLs) may also be bound by gp120. Lipid rafts, enriched with GSLs and cholesterol, are required for HIV entry and therefore the binding of gp120 to GSL-containing vesicles has been studied. Most of the GSL-structures were within the theoretical raft fraction on a discontinuous sucrose gradient while gp120 binding occurred outside of this fraction where a minority of structures migrated. Gb3 fatty acid content modulated binding. Gp120 bound preferentially to structures depleted of cholesterol and binding was enhanced by treating gp120 with CD4. Two water-soluble mimics of Gb3 inhibited gp120 binding to the different structures. The results demonstrate that the aglycone modulation of GSLs alters their receptor function and that the soluble mimics inhibit binding.

Many crucial cellular processes take place at the plasma membrane. The latter is a complex, two-dimensional medium exhibiting significant lateral structure. As a result, a number of non-classical processes, including anomalous diffusion, compartimentalisation and fractal kinetics take place at the membrane surface. The evaluation of various hypotheses and theories about the membrane is currently very difficult because no general modelling framework is available. In this thesis, we present a stochastic, spatially explicit Monte Carlo model for the plasma membrane that accounts for illmixedness, mobile lipid microdomains, fixed proteins, cytoskeletal fence structures and other interactions. We interrogate this model to obtain three classes of results, regarding (1) the effect of lipid microdomains on protein dynamics on the membrane (2) the effects of microdomains, cytoskeletal fences and fixed proteins on the nature of the (anomalous) diffusion on the membrane and (3) the effects of obstructed diffusion on reaction kinetics at the membrane. We find that the presence of lipid microdomains can lead to nonclassical phenomena such as increased collision rates and differences between long-range and short-range diffusion coefficients. Our results also suggest that experimental techniques measuring long-range diffusion may not be sufficiently discriminating and hence cannot be used to infer quantitative information about the presence and characteristics of microdomains. With regard to anomalous diffusion in particular, we find that to explain this phenomenon at the levels observed in vivo, a number of interactions are required, including (but not necessarily limited to) obstacle-induced diffusion and segregation, or exclusion from microdomains. The effects of these different interactions upon the nature of the diffusion appear to be approximately additive. Finally, we show that a widely used non-spatial method, the Stochastic Simulation Algorithm, can be modified to take into account anomalous diffusion and that this significantly increases its predictive accuracy. The model presented in this thesis is expected to be of future value in evaluating different models of cell surface processes.

Many crucial cellular processes take place at the plasma membrane. The latter is a complex, two-dimensional medium exhibiting significant lateral structure. As a result, a number of non-classical processes, including anomalous diffusion, compartimentalisation and fractal kinetics take place at the membrane surface. The evaluation of various hypotheses and theories about the membrane is currently very difficult because no general modelling framework is available. In this thesis, we present a stochastic, spatially explicit Monte Carlo model for the plasma membrane that accounts for illmixedness, mobile lipid microdomains, fixed proteins, cytoskeletal fence structures and other interactions. We interrogate this model to obtain three classes of results, regarding (1) the effect of lipid microdomains on protein dynamics on the membrane (2) the effects of microdomains, cytoskeletal fences and fixed proteins on the nature of the (anomalous) diffusion on the membrane and (3) the effects of obstructed diffusion on reaction kinetics at the membrane. We find that the presence of lipid microdomains can lead to nonclassical phenomena such as increased collision rates and differences between long-range and short-range diffusion coefficients. Our results also suggest that experimental techniques measuring long-range diffusion may not be sufficiently discriminating and hence cannot be used to infer quantitative information about the presence and characteristics of microdomains. With regard to anomalous diffusion in particular, we find that to explain this phenomenon at the levels observed in vivo, a number of interactions are required, including (but not necessarily limited to) obstacle-induced diffusion and segregation, or exclusion from microdomains. The effects of these different interactions upon the nature of the diffusion appear to be approximately additive. Finally, we show that a widely used non-spatial method, the Stochastic Simulation Algorithm, can be modified to take into account anomalous diffusion and that this significantly increases its predictive accuracy. The model presented in this thesis is expected to be of future value in evaluating different models of cell surface processes.

Many crucial cellular processes take place at the plasma membrane. The latter is a complex, two-dimensional medium exhibiting significant lateral structure. As a result, a number of non-classical processes, including anomalous diffusion, compartimentalisation and fractal kinetics take place at the membrane surface. The evaluation of various hypotheses and theories about the membrane is currently very difficult because no general modelling framework is available. In this thesis, we present a stochastic, spatially explicit Monte Carlo model for the plasma membrane that accounts for illmixedness, mobile lipid microdomains, fixed proteins, cytoskeletal fence structures and other interactions. We interrogate this model to obtain three classes of results, regarding (1) the effect of lipid microdomains on protein dynamics on the membrane (2) the effects of microdomains, cytoskeletal fences and fixed proteins on the nature of the (anomalous) diffusion on the membrane and (3) the effects of obstructed diffusion on reaction kinetics at the membrane. We find that the presence of lipid microdomains can lead to nonclassical phenomena such as increased collision rates and differences between long-range and short-range diffusion coefficients. Our results also suggest that experimental techniques measuring long-range diffusion may not be sufficiently discriminating and hence cannot be used to infer quantitative information about the presence and characteristics of microdomains. With regard to anomalous diffusion in particular, we find that to explain this phenomenon at the levels observed in vivo, a number of interactions are required, including (but not necessarily limited to) obstacle-induced diffusion and segregation, or exclusion from microdomains. The effects of these different interactions upon the nature of the diffusion appear to be approximately additive. Finally, we show that a widely used non-spatial method, the Stochastic Simulation Algorithm, can be modified to take into account anomalous diffusion and that this significantly increases its predictive accuracy. The model presented in this thesis is expected to be of future value in evaluating different models of cell surface processes.

Many crucial cellular processes take place at the plasma membrane. The latter is a complex, two-dimensional medium exhibiting significant lateral structure. As a result, a number of non-classical processes, including anomalous diffusion, compartimentalisation and fractal kinetics take place at the membrane surface. The evaluation of various hypotheses and theories about the membrane is currently very difficult because no general modelling framework is available. In this thesis, we present a stochastic, spatially explicit Monte Carlo model for the plasma membrane that accounts for illmixedness, mobile lipid microdomains, fixed proteins, cytoskeletal fence structures and other interactions. We interrogate this model to obtain three classes of results, regarding (1) the effect of lipid microdomains on protein dynamics on the membrane (2) the effects of microdomains, cytoskeletal fences and fixed proteins on the nature of the (anomalous) diffusion on the membrane and (3) the effects of obstructed diffusion on reaction kinetics at the membrane. We find that the presence of lipid microdomains can lead to nonclassical phenomena such as increased collision rates and differences between long-range and short-range diffusion coefficients. Our results also suggest that experimental techniques measuring long-range diffusion may not be sufficiently discriminating and hence cannot be used to infer quantitative information about the presence and characteristics of microdomains. With regard to anomalous diffusion in particular, we find that to explain this phenomenon at the levels observed in vivo, a number of interactions are required, including (but not necessarily limited to) obstacle-induced diffusion and segregation, or exclusion from microdomains. The effects of these different interactions upon the nature of the diffusion appear to be approximately additive. Finally, we show that a widely used non-spatial method, the Stochastic Simulation Algorithm, can be modified to take into account anomalous diffusion and that this significantly increases its predictive accuracy. The model presented in this thesis is expected to be of future value in evaluating different models of cell surface processes.

Os mastócitos são células multifuncionais do sistema imunológico que participam em diversos processos biológicos. As funções dos mastócitos estão diretamente relacionados com a sua ativação e, subsequente, liberação de mediadores químicos. Os eventos iniciais da ativação dos mastócitos e da transdução de sinais ocorrem em microdomínios lipídicos (lipid rafts) da membrana plasmática. Os gangliosídeos derivados do GD1b são constituintes dos lipid rafts de mastócitos de roedores. O intercruzamento destes gangliosídeos pelo mAb AA4, resulta na formação de agregados (caps) na superfície celular e promove uma ativação parcial dos mastócitos, sem que ocorra a desgranulação. A ativação é semelhante a observada quando os FcRIs são intercruzados por antígenos multivalentes ligados a IgEs, mas neste caso ocorre a desgranulação. O presente estudo tem como objetivo caracterizar o papel dos gangliosídeos derivados do GD1b na liberação de mediadores de mastócitos da linhagem RBL-2H3. O intercruzamento dos gangliosídeos derivados do GD1b resulta na ativação dos fatores de transcrição NFAT e NFB e esta ativação é mediada pela proteína quinase Syk. A ativação destes fatores de transcrição resulta na liberação de mediadores neo-sintetizados, tais como: TNF-, interleucina (IL)-4. Por outro lado, o intercruzamento dos gangliosídeos derivados de GD1b não induz a liberação dos mediadores neoformados como o leucotrieno B4 (LTB4) e o leucotrieno C4 (LTC4). A agregação dos gangliosídeos derivados do GD1b resulta na desorganização dos lipid rafts e na redistribuição de seus componentes, como demostrado pela análise proteômica. Estes dados mostraram proteínas capazes de desencadear uma ativação parcial dos mastócitos e proteínas reguladoras negativas da desgranulação estão up reguladas, enquanto que proteínas críticas para a transdução do sinal estão down reguladas. Os resultados obtidos neste trabalho demonstram que os gangliosídeos derivados do GD1b desempenham papel crucial na integridade dos lipid rafts modulando a ativação e liberação de mediadores de mastócitos. / Mast cells are immunoregulatory cells that participate in diverse biological events. The action of mast cells is directly related to their activation and subsequent mediator release. Early signal transduction events occur in lipid rafts in the plasma membrane. GD1b-derived gangliosides are known constituents of lipid rafts in rodent mast cells. The cross-linking of these gangliosides by mAb AA4 results in a partial activation of mast cells similar to that observed when FcRIs are cross-linked, but does not result in the mast cell degranulation. With time, the gangliosides bound to mAb AA4 cap on the cell surface. The present study aims to characterize the role of the rodent mast cell specific gangliosides derived from GD1b in mediator release from RBL-2H3 mast cells. Cross-linking the GD1b-derived gangliosides activated the transcription factors NFAT and NFB and this activation was mediated by Syk. The activation of theses transcription factors by cross-linked GD1b-derived gangliosides results in the release of the neo-synthesized mediators TNF- and interleukin (IL)-4. However, cross-linking GD1b-derived gangliosides did not stimulate release of the newly formed mediators leukotriene B4 (LTB4) and leukotriene C4 (LTC4). Capping of GD1b-derived gangliosides disorganized lipid rafts and resulted in a redistribution of lipid raft components. Proteomic analysis showed that proteins that trigger mast cell activation and negative regulatory proteins of degranulation are up regulated, whereas proteins critical for signal transduction are down regulated in mast cells where the gangliosides are capped. The results of this work demonstrate that the mast cell-specific GD1b-derived gangliosides are crucial in maintaining the functional integrity of the lipid rafts and modulate cell activation and subsequent mediator release from mast cells.

A organização da membrana biológica em microdomínios tem um papel chave em vários processos celulares semelhante a receptores protéicos e a transdução de sinal. A existência de microdomínios, também denominados de rafts tem sido explicada pela separação das membranas lipídicas em duas fases: liquida cristalina (L) e fase liquida ordenada (Lo) rica em colesterol e esfingolipídeos. Assim, o enfoque deste projeto foi correlacionar mecanismos de controle da atividade da fosfatase alcalina (TNAP) com a organização intermolecular e o estado de fase de alguns lipídios que compõem as vesículas da matrix. Foi estudada a modulação da atividade da enzima e sua inserção à sistemas de lipossomos constituídos com diferentes composições lipídicas (Dipalmitoilfosfatidilcolina, Colesterol, Esfingomielina e Gangliosídeo) como um mecanismo de regulação e transdução entre enzimas que não compartilham intermediários metabólicos comuns. Isto é, verificar como mudanças de organização molecular, induzida por colesterol e/ou outros lipídios, podem modular a atividade de enzimas regulando a produção de mensageiros lipídicos secundários e/ou processos de fusão e recombinação topológica da bicamada lipídica, modulando concomitantemente a atividade da fosfatase alcalina. Com tal propósito, a TNAP foi reconstituída em lipossomos constituídos de DPPC e lipossomos mistos formando sistemas binários DPPC:Chol, DPPC:SM e DPPC:GM1 com razões molares de (9:1); sistemas terciários DPPC:Chol:SM, DPPC:Chol:GM1 e DPPC:SM:GM1 com razões molares de (8:1:1) e por fim sistemas quaternários constituídos de DPPC:Chol:SM:GM1 (7:1:1:1). Estes sistemas foram propostos com o intuito de mimetizarmos os lipid rafts existentes nas membranas biológicas, porém utilizando lipídios que já foram identificados e quantificados nas vesículas da matrix. Foram avaliados os efeitos da composição lipídica dos lipossomos na inserção da enzima aos sistemas vesiculares. Além disso, foram realizados estudos biofísicos de calorimetria analisando como os parâmetros termodinâmicos são afetados com as diferentes composições lipídicas e pela presença da enzima ancorada aos sistemas. A reconstituição da enzima a lipossomos constituídos de DPPC proporcionou uma incorporação em torno de 80% da atividade enzimática. Estudos termodinâmicos dos proteolipossomos formados evidenciaram uma queda significativa nos valores de variação de entalpia em relação aos sistemas de lipossomos (de 7,63 a 1,88 kcal.mol-1). Lipossomos binários constituídos de DPPC:Chol em concentrações crescentes (9:1, 9:2, 9:3, 7:3, 9:4 e 9:5 razão molar) foram estudados tanto pelos parâmetros biofísicos como pela habilidade de inserção da enzima a tais sistemas. Foi observado um significativo decréscimo nos valores de variação entalpia com o aumento da proporção de colesterol no lipossomo. Além disso, a presença do colesterol proporcionou uma redução na inserção da atividade catalítica em até 42%, quando utilizada a composição lipídica de 9:5 DPPC:Chol. Dos sistemas binários formados com razões molares 9:1, o que apresentou maior porcentagem de reconstituição da TNAP foi o sistemas DPPC:Chol, apresentando em torno de 62% de incorporação da enzima. Os sistemas terciários apresentaram ao redor de 30% de incorporação da atividade catalítica e o sistema quaternário em torno de 25%. Além dos ensaios de atividade enzimática, a incorporação da enzima aos sistemas vesiculares também pôde ser comprovada pelas mudanças nos parâmetros termodinâmicas detectados por DSC. Nos estudos de calorimetria de todos os sistemas de proteolipossomos formados, foram observadas significativas diminuições nos valores de variação de entalpia quando comparados aos sistemas de lipossomos correspondentes. Deste modo, os resultados aqui apresentados fornecem novas informações que poderão contribuir tanto para a compreensão do comportamento da atividade da fosfatase alcalina na presença de diferentes composições lipídicas dos microdomínios existente membrana, quanto para o entendimento dos processos de regulação da enzima durante o processo de biomineralização. / The organization of the biological membrane in microdomains has a key roll in many cellular processes similar to proteic receptors and signal transduction. The existence of microdomains, also called rafts, has been explained by the lipid membrane separation in two phases: crystalline phase (L) and ordinate liquid phase (Lo), rich in cholesterol and sphingolipids. The focus of this Project was to correlate activity control mechanisms of the alkaline phosphatase (TNAP) with the intermolecular organization and the phase stat of some lipids that comprise the matrix vesicles. The enzyme activity modulation and its insertion into liposomes systems, constituted by different lipid compositions (DPPC, Chol, SM e GM1) as a regulation and transduction mechanism between enzymes that do not share common intermediary metabolites, was studied. That is, to verify how molecular organization changes, induced by cholesterol and/or other lipids, can modulate the enzyme activity regulating the production of secondary lipid messengers and/or fusion processes and topological recombination of the lipidic bilayer, concomitantly modeling the alkaline phosphatase activity. TNAP was then reconstituted in liposomes constituted by DPPC and mixed liposomes forming binary systems DPPC:Chol , DPPC:SM , DPPC: Chol:GM1 with (9:1) molar rates; tertiary systems DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 with (8:1:1) molar rates and finally quaternary system constituted by DPPC:Chol:SM:GM1 (7:1:1:1). These systems were proposed aiming the mimetization of lipid rafts existent in biological membranes, but using lipids that had already been identified and quantified in the matrix vesicles. The effects of liposome lipid composition in the enzyme insertion to the vesicular systems were assayed. Besides that, calorimetry biophysical studies were done analyzing how the thermodynamic parameters are affected by the different lipid compositions e by the presence of the systems anchored enzyme. The enzyme reconstruction to the DPPC constituted liposomes has provided an incorporation of around 80% of the enzyme activity. Thermodynamic studies of the proteoliposomes formed have shown a significant decrease in the H values in relation to the liposomes systems (from 7.63 to 1.88 kcal.mol-1). Binary liposomes constituted of DPPC:Chol in increasing concentrations (9:1, 9:2, 9:3, 7:3, 9:4 e 9:5 molar ratio) were studied by the biophysical parameters as well as by the insertion ability of the enzyme into those systems. A significant decrease in the enthalpy values with the increase of the cholesterol proportion in the liposome was observed. Besides that, the presence of cholesterol has allowed a reduction in the insertion of the catalytic activity in up to 42% when the lipid composition 9:5 DPPPC:Chol was used. Among the binary systems formed with molar ratios of 9:1, the one which showed the highest percentage of TNAP reconstitution was the DPPC:Chol system, with around 62% enzyme incorporation. The tertiary systems had around 30% incorporation of the catalytic activity, and the quaternary system around 25%. Besides the enzymatic activity assays, the enzyme incorporation to the vesicular systems can also be verified by the thermodynamic parameters change detected by DSC. In the calorimetry studies of all the proteoliposomes formed, significant decreases in the enthalpy values were observed when compared to the corresponding liposomes systems. Thereby, the results presented here provide new information that can contribute to understand the alkaline phosphatase behavior in the presence of different microdomain lipid compositions existent in the membrane, as well as understanding the regulation processes of the enzyme during the biomineralization process.

Os mastócitos são células multifuncionais do sistema imunológico que participam em diversos processos biológicos. As funções dos mastócitos estão diretamente relacionados com a sua ativação e, subsequente, liberação de mediadores químicos. Os eventos iniciais da ativação dos mastócitos e da transdução de sinais ocorrem em microdomínios lipídicos (lipid rafts) da membrana plasmática. Os gangliosídeos derivados do GD1b são constituintes dos lipid rafts de mastócitos de roedores. O intercruzamento destes gangliosídeos pelo mAb AA4, resulta na formação de agregados (caps) na superfície celular e promove uma ativação parcial dos mastócitos, sem que ocorra a desgranulação. A ativação é semelhante a observada quando os FcRIs são intercruzados por antígenos multivalentes ligados a IgEs, mas neste caso ocorre a desgranulação. O presente estudo tem como objetivo caracterizar o papel dos gangliosídeos derivados do GD1b na liberação de mediadores de mastócitos da linhagem RBL-2H3. O intercruzamento dos gangliosídeos derivados do GD1b resulta na ativação dos fatores de transcrição NFAT e NFB e esta ativação é mediada pela proteína quinase Syk. A ativação destes fatores de transcrição resulta na liberação de mediadores neo-sintetizados, tais como: TNF-, interleucina (IL)-4. Por outro lado, o intercruzamento dos gangliosídeos derivados de GD1b não induz a liberação dos mediadores neoformados como o leucotrieno B4 (LTB4) e o leucotrieno C4 (LTC4). A agregação dos gangliosídeos derivados do GD1b resulta na desorganização dos lipid rafts e na redistribuição de seus componentes, como demostrado pela análise proteômica. Estes dados mostraram proteínas capazes de desencadear uma ativação parcial dos mastócitos e proteínas reguladoras negativas da desgranulação estão up reguladas, enquanto que proteínas críticas para a transdução do sinal estão down reguladas. Os resultados obtidos neste trabalho demonstram que os gangliosídeos derivados do GD1b desempenham papel crucial na integridade dos lipid rafts modulando a ativação e liberação de mediadores de mastócitos. / Mast cells are immunoregulatory cells that participate in diverse biological events. The action of mast cells is directly related to their activation and subsequent mediator release. Early signal transduction events occur in lipid rafts in the plasma membrane. GD1b-derived gangliosides are known constituents of lipid rafts in rodent mast cells. The cross-linking of these gangliosides by mAb AA4 results in a partial activation of mast cells similar to that observed when FcRIs are cross-linked, but does not result in the mast cell degranulation. With time, the gangliosides bound to mAb AA4 cap on the cell surface. The present study aims to characterize the role of the rodent mast cell specific gangliosides derived from GD1b in mediator release from RBL-2H3 mast cells. Cross-linking the GD1b-derived gangliosides activated the transcription factors NFAT and NFB and this activation was mediated by Syk. The activation of theses transcription factors by cross-linked GD1b-derived gangliosides results in the release of the neo-synthesized mediators TNF- and interleukin (IL)-4. However, cross-linking GD1b-derived gangliosides did not stimulate release of the newly formed mediators leukotriene B4 (LTB4) and leukotriene C4 (LTC4). Capping of GD1b-derived gangliosides disorganized lipid rafts and resulted in a redistribution of lipid raft components. Proteomic analysis showed that proteins that trigger mast cell activation and negative regulatory proteins of degranulation are up regulated, whereas proteins critical for signal transduction are down regulated in mast cells where the gangliosides are capped. The results of this work demonstrate that the mast cell-specific GD1b-derived gangliosides are crucial in maintaining the functional integrity of the lipid rafts and modulate cell activation and subsequent mediator release from mast cells.

A organização da membrana biológica em microdomínios tem um papel chave em vários processos celulares semelhante a receptores protéicos e a transdução de sinal. A existência de microdomínios, também denominados de rafts tem sido explicada pela separação das membranas lipídicas em duas fases: liquida cristalina (L) e fase liquida ordenada (Lo) rica em colesterol e esfingolipídeos. Assim, o enfoque deste projeto foi correlacionar mecanismos de controle da atividade da fosfatase alcalina (TNAP) com a organização intermolecular e o estado de fase de alguns lipídios que compõem as vesículas da matrix. Foi estudada a modulação da atividade da enzima e sua inserção à sistemas de lipossomos constituídos com diferentes composições lipídicas (Dipalmitoilfosfatidilcolina, Colesterol, Esfingomielina e Gangliosídeo) como um mecanismo de regulação e transdução entre enzimas que não compartilham intermediários metabólicos comuns. Isto é, verificar como mudanças de organização molecular, induzida por colesterol e/ou outros lipídios, podem modular a atividade de enzimas regulando a produção de mensageiros lipídicos secundários e/ou processos de fusão e recombinação topológica da bicamada lipídica, modulando concomitantemente a atividade da fosfatase alcalina. Com tal propósito, a TNAP foi reconstituída em lipossomos constituídos de DPPC e lipossomos mistos formando sistemas binários DPPC:Chol, DPPC:SM e DPPC:GM1 com razões molares de (9:1); sistemas terciários DPPC:Chol:SM, DPPC:Chol:GM1 e DPPC:SM:GM1 com razões molares de (8:1:1) e por fim sistemas quaternários constituídos de DPPC:Chol:SM:GM1 (7:1:1:1). Estes sistemas foram propostos com o intuito de mimetizarmos os lipid rafts existentes nas membranas biológicas, porém utilizando lipídios que já foram identificados e quantificados nas vesículas da matrix. Foram avaliados os efeitos da composição lipídica dos lipossomos na inserção da enzima aos sistemas vesiculares. Além disso, foram realizados estudos biofísicos de calorimetria analisando como os parâmetros termodinâmicos são afetados com as diferentes composições lipídicas e pela presença da enzima ancorada aos sistemas. A reconstituição da enzima a lipossomos constituídos de DPPC proporcionou uma incorporação em torno de 80% da atividade enzimática. Estudos termodinâmicos dos proteolipossomos formados evidenciaram uma queda significativa nos valores de variação de entalpia em relação aos sistemas de lipossomos (de 7,63 a 1,88 kcal.mol-1). Lipossomos binários constituídos de DPPC:Chol em concentrações crescentes (9:1, 9:2, 9:3, 7:3, 9:4 e 9:5 razão molar) foram estudados tanto pelos parâmetros biofísicos como pela habilidade de inserção da enzima a tais sistemas. Foi observado um significativo decréscimo nos valores de variação entalpia com o aumento da proporção de colesterol no lipossomo. Além disso, a presença do colesterol proporcionou uma redução na inserção da atividade catalítica em até 42%, quando utilizada a composição lipídica de 9:5 DPPC:Chol. Dos sistemas binários formados com razões molares 9:1, o que apresentou maior porcentagem de reconstituição da TNAP foi o sistemas DPPC:Chol, apresentando em torno de 62% de incorporação da enzima. Os sistemas terciários apresentaram ao redor de 30% de incorporação da atividade catalítica e o sistema quaternário em torno de 25%. Além dos ensaios de atividade enzimática, a incorporação da enzima aos sistemas vesiculares também pôde ser comprovada pelas mudanças nos parâmetros termodinâmicas detectados por DSC. Nos estudos de calorimetria de todos os sistemas de proteolipossomos formados, foram observadas significativas diminuições nos valores de variação de entalpia quando comparados aos sistemas de lipossomos correspondentes. Deste modo, os resultados aqui apresentados fornecem novas informações que poderão contribuir tanto para a compreensão do comportamento da atividade da fosfatase alcalina na presença de diferentes composições lipídicas dos microdomínios existente membrana, quanto para o entendimento dos processos de regulação da enzima durante o processo de biomineralização. / The organization of the biological membrane in microdomains has a key roll in many cellular processes similar to proteic receptors and signal transduction. The existence of microdomains, also called rafts, has been explained by the lipid membrane separation in two phases: crystalline phase (L) and ordinate liquid phase (Lo), rich in cholesterol and sphingolipids. The focus of this Project was to correlate activity control mechanisms of the alkaline phosphatase (TNAP) with the intermolecular organization and the phase stat of some lipids that comprise the matrix vesicles. The enzyme activity modulation and its insertion into liposomes systems, constituted by different lipid compositions (DPPC, Chol, SM e GM1) as a regulation and transduction mechanism between enzymes that do not share common intermediary metabolites, was studied. That is, to verify how molecular organization changes, induced by cholesterol and/or other lipids, can modulate the enzyme activity regulating the production of secondary lipid messengers and/or fusion processes and topological recombination of the lipidic bilayer, concomitantly modeling the alkaline phosphatase activity. TNAP was then reconstituted in liposomes constituted by DPPC and mixed liposomes forming binary systems DPPC:Chol , DPPC:SM , DPPC: Chol:GM1 with (9:1) molar rates; tertiary systems DPPC:Chol:SM, DPPC:Chol:GM1 and DPPC:SM:GM1 with (8:1:1) molar rates and finally quaternary system constituted by DPPC:Chol:SM:GM1 (7:1:1:1). These systems were proposed aiming the mimetization of lipid rafts existent in biological membranes, but using lipids that had already been identified and quantified in the matrix vesicles. The effects of liposome lipid composition in the enzyme insertion to the vesicular systems were assayed. Besides that, calorimetry biophysical studies were done analyzing how the thermodynamic parameters are affected by the different lipid compositions e by the presence of the systems anchored enzyme. The enzyme reconstruction to the DPPC constituted liposomes has provided an incorporation of around 80% of the enzyme activity. Thermodynamic studies of the proteoliposomes formed have shown a significant decrease in the H values in relation to the liposomes systems (from 7.63 to 1.88 kcal.mol-1). Binary liposomes constituted of DPPC:Chol in increasing concentrations (9:1, 9:2, 9:3, 7:3, 9:4 e 9:5 molar ratio) were studied by the biophysical parameters as well as by the insertion ability of the enzyme into those systems. A significant decrease in the enthalpy values with the increase of the cholesterol proportion in the liposome was observed. Besides that, the presence of cholesterol has allowed a reduction in the insertion of the catalytic activity in up to 42% when the lipid composition 9:5 DPPPC:Chol was used. Among the binary systems formed with molar ratios of 9:1, the one which showed the highest percentage of TNAP reconstitution was the DPPC:Chol system, with around 62% enzyme incorporation. The tertiary systems had around 30% incorporation of the catalytic activity, and the quaternary system around 25%. Besides the enzymatic activity assays, the enzyme incorporation to the vesicular systems can also be verified by the thermodynamic parameters change detected by DSC. In the calorimetry studies of all the proteoliposomes formed, significant decreases in the enthalpy values were observed when compared to the corresponding liposomes systems. Thereby, the results presented here provide new information that can contribute to understand the alkaline phosphatase behavior in the presence of different microdomain lipid compositions existent in the membrane, as well as understanding the regulation processes of the enzyme during the biomineralization process.

Interleukin-1-beta (IL-1β) is a potent proinflammatory cytokine. A primary outcome of IL-1β signaling is the activation of NFκB, a transcription factor that induces a large number of immune molecules, apoptotic factors, anti-apoptotic factors, and other transcription factors. Recent work has demonstrated that the activation of NFκB involves a multistep redox-signaling cascade that requires endocytosis of the interleukin receptor (IL-1R1)/ligand pair and superoxide production by NADPH oxidase 2 (Nox2) within the resulting newly formed early endosome. Hydrogen peroxide produced by the rapid dismutation of superoxide is necessary for the subsequent downstream recruitment of IL-1R1 effectors (TRAF6, IKK kinases) and ultimately the activation of NFκB. In this thesis, I have further dissected the spatial and temporal events that coordinate signaling processes of the IL-1β pathway. Using a combination of biophotonic imaging, immunofluorescence imaging, and lipid raft density gradient isolation, I demonstrate that both Nox2 and IL-1R1 are constitutively present in lipid raft microdomains on the plasma membrane. Stimulation by IL-1β induces endocytosis of Nox2 and IL-1R1 from the plasma membrane into caveolin-1, lipid raft positive early endosomes. Further, inhibition of lipid raft mediated endocytosis or deletion of caveolin-1 inhibits activation of NFκB, by IL-1β. We have also identified Vav1 as the Rac1 guanine exchange factor that is recruited to caveolin-1 positive lipid rafts following IL-1β stimulation, and demonstrated that dominant negative Vav1 inhibits NFκB activation by IL-1β. Following this work, I utilized assays for redox sensitivity and mass spectrometry to demonstrate that C70, C73, and C105 are hydrogen peroxide sensitive cysteines within the RING domain of TRAF6. I further demonstrate that hydrogen peroxide does not alter the E3 ubiquitin ligase activity associated with the TRAF6 RING domain. My findings suggest that the redox sensitivity of the RING domain mediates TRAF6 recruitment to the receptor complex. This is supported by the observation that hydrogen peroxide treatment of TRAF6, but not early signaling effectors (IL-1R1, IRAK1, IRAK4, MyD88) mediates TRAF6 recruitment to the IL-1 receptor complex. Further, mutation of the identified redox sensitive cysteines inhibits IL-1β signaling and NFκB activation. This research has helped to refine the understanding of the IL-1β signaling pathway, and may ultimately lead to new therapeutic targets for controlling inflammation.