Closed-loop combustion control is an on-going field of research for improving reducing engine emissions and increasing efficiency. Cylinder pressure is a key parameter to monitor for combustion feedback. Measuring pressure with a transducer is an option, although being able to estimate the pressure based on the crank angle measurement instead would be beneficial in terms of costs. A virtual crank angle based pressure sensor was therefore developed within this thesis. It was studied how the in-cylinder pressure trace for a full closed cycle could be modelled from a pressure trace from a rigid crankshaft model, the angular velocity measurement and heat release modelling. The pressure trace from the crankshaft model was subjected to a singularity at TDC and torsional oscillations, it was therefore of interest to study whether the singularity could be avoided by modelling the heat release. Further on, the indicated work and total heat released during combustion were estimated from the angular velocity measurements as they are important parameters for determining the heat release trace.   It was found that the indicated work could be approximated by comparing the kinetic power trace, obtained from the measured angular velocity, with the piston power trace, estimated using isentropic pressure curves for the compression and expansion within the cylinder. Accurate results were obtained for operating points at 800 rpm while large deviations were seen for higher speeds as a consequence of larger torsional effect on the angular velocity trace; on the form of perturbed oscillations. The results could be improved from local averaging of the kinetic power trace at the occasions of deceleration, although it could be concluded that only the low speed operating were still accurate enough.   The kinetic power trace was attempted to be corrected for torsional power using angular displacement estimations of the crankshaft nodes from a dynamic crankshaft model. Even though the model seemed to capture the torsional behaviour at parts of the cycle, the oscillations could not be completely removed and it was determined that the final work estimate could not be improved from the torsional power estimate. The torsion was further studied regarding frequency and amplitude of the oscillations within the angular velocity and acceleration trace. No clear relations between the torsional behaviour and operating speed and load could be concluded. Further, since inversion of the dynamic crankshaft model for pressure estimation resulted in an improper solution since before, the model was iterated instead. The pressure trace could thereby be derived accounting for torsion, however the trace still contained oscillations which highlights the challenge of estimating the torsion accurately. The torsion is a complex phenomenon to describe and further development of a model for estimating the torsion with high accuracy for all operating points would improve the virtual pressure sensor significantly.   The heat release was, as a first step, modelled as isochoric and isobaric. These models gave information of the limits of SOC by comparing the indicated work from the resulting pressure trace with the work estimate from the angular velocity measurement. Further, one Wiebe function was parametrised such that the resulting pressure derivative during late combustion was adapted to the trace from the crankshaft model in a least-square sense. This allowed for better adaption as the partial pressure trace was subjected to torsional oscillations. The fitted Wiebe function described the diffusive combustion well but missed out the shape of the premixed combustion. Lastly, a double Wiebe function parametrisation was done where the diffusive combustion function was fitted to the late combustion data and the premixed combustion function was adapted such that the resulting indicated work matched the estimated work. To receive more accurate results, the premixed SOC and duration had to be approximated beforehand from the kinetic power trace. The virtual pressure sensor and most of the sub models were most accurate for low speed operating points. It was concluded that the reason is most probably the torsional effect on the input data to all sub models. It was shown that the crankshaft model can be complemented with heat release estimations which improved the final pressure trace and removed the singularity present around TDC. / Förbränningsåterkoppling är ett aktuellt forskningsområde inom utvecklingsarbetet för att minska utsläpp och öka verkningsgraden hos förbränningsmotorer. Cylindertryck är en viktig parameter att mäta . Ett alternativ är att använda en tryckgivare men det skulle vara mer kostnadseffektivt att kunna uppskatta trycket baserat på vevvinkeln som redan idag mäts i motorer. Därav utvecklades en virtuell sensor för uppskattning av cylindertrycket genom detta examensarbete. Studien har berört hur tryck spår, bitvis noggranna för kompressionen och expansionen, från en stel vevaxelmodell kan kompletteras med modeller för värmeavgivningen från förbränningen för att erhålla ett fullt tryck spår. För att kunna bygga och utveckla modellerna utvecklades en metod för att bestämma det indikerade arbetet baserat på den uppmätta varvtalssignalen som beror mycket på hur förbränningen skett och är därmed en viktig parameter vid modellerande av värmeavgivningen.   Det indikerade arbetet kunde uppskattas genom att jämföra den kinetiska effekten med den effekt som kolvarna totalt bidrog med. Det upptäcktes att offseten mellan kurvorna motsvarade effekten av förlusterna och lasten som därmed kunde bestämmas vid de punkter där momentet från cylindrarna var i jämvikt. Den kinetiska effekten beräknades från varvtalssignalen medan effekten från kolvarna uppskattades genom att använda isentropiska tryckkurvor för kompressionen och expansionen, innan och efter förbränningen respektive. Relativt noggranna resultat erhölls för arbetspunkterna med ett varvtal på 800 rpm medan större avvikelser inträdde vid högre varvtal. Anledningen till detta var att torsionssvängningar influerade varvtalssignalen mer vid högre varvtal. Resultaten kunde förbättras genom lokal medelvärdesbildning av den kinetiska effekten vid de decelerationer som sker efter förbränningen i respektive cylinder. II   Torsionens inverkan på vevaxelns dynamik uppskattades genom att använda estimeringar av förvridningen av vevaxeln från en dynamisk vevaxelmodell. Uppskattningen tycktes vara tillräckligt noggrann inom vissa intervall men det var inte möjligt att avlägsna torsionssvängningarna i kinetiska effektspåret för hela cykeln. Uppskattningen av indikerat arbete kunde därför inte förbättras genom denna torsionsuppskattning. Torsionen var vidare studerad i form av frekvens och amplitud av svängningarna inom varvtalssignalen. Inga tydliga samband kunde säkerställas mellan svängningarna och arbetspunkternas varvtal och last. Detta tyder på att torsionen är för komplex att förutse. Vidare, då invertering av den dynamiska vevaxelmodellen tidigare visat sig ge en oriktig lösning kunde modellen istället itereras för att bestämma tryck spåret likt tidigare gjort för den stela vevaxelmodellen. Torsionssvängningarna influerade dock fortfarande det resulterande tryck spåret. Det finns stor potential att förbättra den virtuella sensorn om torsionen kan uppskattas noggrant för alla arbetspunkter.   Värmeavgivningen från förbränning var först modellerad som isochorisk och isobarisk i två respektive modeller. Dessa modeller gav information om gränsvärdena för tändningen genom att finna den tändning för modellerna som resulterade i samma arbete som det tidigare estimerade indikerade arbetet. Därefter anpassades en Wiebe funktion så att den resulterande tryckderivatan minsta-kvadrat anpassades till tryckderivatan från vevaxelmodellen under den sena förbränningen där vevaxelmodellen var mest noggrann. Wiebe funktion gav en bra anpassning till den senare diffusiva förbränningen men var inte tillräcklig för att beskriva den förblandade förbränningen. Slutligen anpassades två Wiebe funktioner där den diffusiva förbränningen anpassades likt för singel Wiebe-funktions anpassningen medan den förblandade förbränningen anpassades så att det resulterande arbetet stämde med det uppskattade indikerade arbetet. För att få bättre resultat bestämdes den förblandade förbränningens start och duration från uppskattningen av den kinetiska effekten innan anpassningen.   Den virtuella trycksensorn och de flesta av dess delmodeller var mest noggranna för arbetspunkterna vid låga varvtal. Slutsatsen var att det var främst på grund av torsionssvängningarnas påverkan på insignalerna till delmodellerna som noggrannheten föll för de högra varvtalen. Genom denna studie visades det att deltrycksspåret från vevaxelmodellen kunde kompletteras med en modell för värmeavgivningen för att slutligen få en bättre uppskattning av hela tryck spåret där singulariteten vid TDC kunde undvikas.

Closed-loop combustion control is an on-going field of research for improving reducing engine emissions and increasing efficiency. Cylinder pressure is a key parameter to monitor for combustion feedback. Measuring pressure with a transducer is an option, although being able to estimate the pressure based on the crank angle measurement instead would be beneficial in terms of costs. A virtual crank angle based pressure sensor was therefore developed within this thesis. It was studied how the in-cylinder pressure trace for a full closed cycle could be modelled from a pressure trace from a rigid crankshaft model, the angular velocity measurement and heat release modelling. The pressure trace from the crankshaft model was subjected to a singularity at TDC and torsional oscillations, it was therefore of interest to study whether the singularity could be avoided by modelling the heat release. Further on, the indicated work and total heat released during combustion were estimated from the angular velocity measurements as they are important parameters for determining the heat release trace. It was found that the indicated work could be approximated by comparing the kinetic power trace, obtained from the measured angular velocity, with the piston power trace, estimated using isentropic pressure curves for the compression and expansion within the cylinder. Accurate results were obtained for operating points at 800 rpm while large deviations were seen for higher speeds as a consequence of larger torsional effect on the angular velocity trace; on the form of perturbed oscillations. The results could be improved from local averaging of the kinetic power trace at the occasions of deceleration, although it could be concluded that only the low speed operating were still accurate enough. The kinetic power trace was attempted to be corrected for torsional power using angular displacement estimations of the crankshaft nodes from a dynamic crankshaft model. Even though the model seemed to capture the torsional behaviour at parts of the cycle, the oscillations could not be completely removed and it was determined that the final work estimate could not be improved from the torsional power estimate. The torsion was further studied regarding frequency and amplitude of the oscillations within the angular velocity and acceleration trace. No clear relations between the torsional behaviour and operating speed and load could be concluded. Further, since inversion of the dynamic crankshaft model for pressure estimation resulted in an improper solution since before, the model was iterated instead. The pressure trace could thereby be derived accounting for torsion, however the trace still contained oscillations which highlights the challenge of estimating the torsion accurately. The torsion is a complex phenomenon to describe and further development of a model for estimating the torsion with high accuracy for all operating points would improve the virtual pressure sensor significantly. The heat release was, as a first step, modelled as isochoric and isobaric. These models gave information of the limits of SOC by comparing the indicated work from the resulting pressure trace with the work estimate from the angular velocity measurement. Further, one Wiebe function was parametrised such that the resulting pressure derivative during late combustion was adapted to the trace from the crankshaft model in a least-square sense. This allowed for better adaption as the partial pressure trace was subjected to torsional oscillations. The fitted Wiebe function described the diffusive combustion well but missed out the shape of the premixed combustion. Lastly, a double Wiebe function parametrisation was done where the diffusive combustion function was fitted to the late combustion data and the premixed combustion function was adapted such that the resulting indicated work matched the estimated work. To receive more accurate results, the premixed SOC and duration had to be approximated beforehand from the kinetic power trace. The virtual pressure sensor and most of the sub models were most accurate for low speed operating points. It was concluded that the reason is most probably the torsional effect on the input data to all sub models. It was shown that the crankshaft model can be complemented with heat release estimations which improved the final pressure trace and removed the singularity present around TDC. / Förbränningsåterkoppling är ett aktuellt forskningsområde inom utvecklingsarbetet för att minska utsläpp och öka verkningsgraden hos förbränningsmotorer. Cylindertryck är en viktig parameter att mäta . Ett alternativ är att använda en tryckgivare men det skulle vara mer kostnadseffektivt att kunna uppskatta trycket baserat på vevvinkeln som redan idag mäts i motorer. Därav utvecklades en virtuell sensor för uppskattning av cylindertrycket genom detta examensarbete. Studien har berört hur tryck spår, bitvis noggranna för kompressionen och expansionen, från en stel vevaxelmodell kan kompletteras med modeller för värmeavgivningen från förbränningen för att erhålla ett fullt tryck spår. För att kunna bygga och utveckla modellerna utvecklades en metod för att bestämma det indikerade arbetet baserat på den uppmätta varvtalssignalen som beror mycket på hur förbränningen skett och är därmed en viktig parameter vid modellerande av värmeavgivningen. Det indikerade arbetet kunde uppskattas genom att jämföra den kinetiska effekten med den effekt som kolvarna totalt bidrog med. Det upptäcktes att offseten mellan kurvorna motsvarade effekten av förlusterna och lasten som därmed kunde bestämmas vid de punkter där momentet från cylindrarna var i jämvikt. Den kinetiska effekten beräknades från varvtalssignalen medan effekten från kolvarna uppskattades genom att använda isentropiska tryckkurvor för kompressionen och expansionen, innan och efter förbränningen respektive. Relativt noggranna resultat erhölls för arbetspunkterna med ett varvtal på 800 rpm medan större avvikelser inträdde vid högre varvtal. Anledningen till detta var att torsionssvängningar influerade varvtalssignalen mer vid högre varvtal. Resultaten kunde förbättras genom lokal medelvärdesbildning av den kinetiska effekten vid de decelerationer som sker efter förbränningen i respektive cylinder. Torsionens inverkan på vevaxelns dynamik uppskattades genom att använda estimeringar av förvridningen av vevaxeln från en dynamisk vevaxelmodell. Uppskattningen tycktes vara tillräckligt noggrann inom vissa intervall men det var inte möjligt att avlägsna torsionssvängningarna i kinetiska effektspåret för hela cykeln. Uppskattningen av indikerat arbete kunde därför inte förbättras genom denna torsionsuppskattning. Torsionen var vidare studerad i form av frekvens och amplitud av svängningarna inom varvtalssignalen. Inga tydliga samband kunde säkerställas mellan svängningarna och arbetspunkternas varvtal och last. Detta tyder på att torsionen är för komplex att förutse. Vidare, då invertering av den dynamiska vevaxelmodellen tidigare visat sig ge en oriktig lösning kunde modellen istället itereras för att bestämma tryck spåret likt tidigare gjort för den stela vevaxelmodellen. Torsionssvängningarna influerade dock fortfarande det resulterande tryck spåret. Det finns stor potential att förbättra den virtuella sensorn om torsionen kan uppskattas noggrant för alla arbetspunkter. Värmeavgivningen från förbränning var först modellerad som isochorisk och isobarisk i två respektive modeller. Dessa modeller gav information om gränsvärdena för tändningen genom att finna den tändning för modellerna som resulterade i samma arbete som det tidigare estimerade indikerade arbetet. Därefter anpassades en Wiebe funktion så att den resulterande tryckderivatan minsta-kvadrat anpassades till tryckderivatan från vevaxelmodellen under den sena förbränningen där vevaxelmodellen var mest noggrann. Wiebe funktion gav en bra anpassning till den senare diffusiva förbränningen men var inte tillräcklig för att beskriva den förblandade förbränningen. Slutligen anpassades två Wiebe funktioner där den diffusiva förbränningen anpassades likt för singel Wiebe-funktions anpassningen medan den förblandade förbränningen anpassades så att det resulterande arbetet stämde med det uppskattade indikerade arbetet. För att få bättre resultat bestämdes den förblandade förbränningens start och duration från uppskattningen av den kinetiska effekten innan anpassningen. Den virtuella trycksensorn och de flesta av dess delmodeller var mest noggranna för arbetspunkterna vid låga varvtal. Slutsatsen var att det var främst på grund av torsionssvängningarnas påverkan på insignalerna till delmodellerna som noggrannheten föll för de högra varvtalen. Genom denna studie visades det att deltrycksspåret från vevaxelmodellen kunde kompletteras med en modell för värmeavgivningen för att slutligen få en bättre uppskattning av hela tryck spåret där singulariteten vid TDC kunde undvikas.

Induction machine; outer rotor; water pump; thermal network; thermal analysis; optimization; electromagnetic model; finite element method; thermal insulation class

For many years, community colleges that chose to offer community college baccalaureate (CCB) programs were looked upon in a negative light (Rice, 2015). However, as the need for specialized baccalaureates within specific fields and job markets have continued to grow (McKee, 2005), CCB programs are becoming more widely accepted throughout the United States. In spite of this paradigm shift, Mississippi is one of the remaining states that have not embraced the idea of CCB programs, in spite of its statistical deficiency in regards to baccalaureate degree holding citizens (Williams, 2010). The focus of this study was to explore the perceptions of community college administrators in Mississippi with regards to the influence of CCB programs to the community college mission of institutions in their state. This study indicates that administrators in Mississippi recognize the benefits of offering CCB programs, but do not want CCB programs to take away from the well-established statewide higher education system through mission creep. Many of the strong position statements received overwhelmingly neutral responses. In contrast, Administrators who chose to give their opinion indicated that they are not familiar with how CCB programs are implemented, and do not believe that Mississippi is ready for CCB programs across the state. However, respondents felt that the community college mission is always evolving, should meet students’ needs, and varies from location to location. The findings also show that administrators are favorable to the piloting of CCB programs at a few (1-2) institutions, even though they believe the programs will take funding away from current programs and do not want community colleges evolving into 4-year institutions. The study also concludes that there is a significant difference between institution size and survey questions #18 and #20. There is also a significant difference between length of time in the community college sector and survey questions #15, #17, and #18.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal. / The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal. / The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.