The thesis is a detailed analysis of a selection of poems on similar themes by the Roman elegists in an attempt to estimate the originality of each poet in his treatment of each theme. The literary history of the themes prior to their occurrence in elegy is also considered. The study opens with a discussion of two "generic" themes, the paraclausithyron and the propempticon ("generic" here being used in the sense of classification in terms of the poems' content). It is demonstrated that while the elegists were certainly aware of the Greek komastic tradition and the many topoi associated with it, they are also distinctively Roman and individualistic in their handling of the theme of the exclusus amator (in particular in their employment of religious language in the address to the door). The section on the propempticon concerns Prop.18 and Ovid Am.2.11, and again it is argued that while both poets were aware of the generic conventions deriving from Greek literature, (those of the "schetliastic propempticon") they have made a traditional form serve their own purposes, Propertius "dramatising" the situation (Cyntbia at 1.8.26 is persuaded not to leave) and Ovid flippantly exaggerating and cleverly manipulating the topoi of the genre. The second chapter focusses on three themes which seem to have strong connections with comedy. The first is the rixa or lovers' quarrel in which violence is inflicted by the one party of the love affair on the other. The girl's violence is enjoyed by her lover because it is interpreted as an indication of her passionate love for him, while the lover's violence is a source of regret to him. Comic precedents for both attitudes are produced. Next under discussion is the soldier-rival, based on the character of the miles gloriosus of comedy but adapted to suit each elegist's purposes. The third comic theme is the affair with the ancilia, found in both Propertius and Tibullus but given a very different treatment by each of them. This is perhaps inspired by the comic situation in which the husband is suspected by his carping wife of having an affair with her ancilla. Three themes frequently occurring in Greek epigram are discussed in the third chapter. First under consideration is the figure of the irrisor amoris, the man who mocked love only to fall in love himself; variations on this theme by Tibullus (l,8.7lff. and 1.2.87ff.) and Propertius (l.9.lff.) are examined in detail. There follows an analysis of Tibullus 1.2.25ff., Prop.3.l6.11ff. and Ovid Am.l.6.13ff., adaptations of the epigrammatic motif of divine protection for the lover when he comes to his girl at night (but extended by Tibullus and Propertius to the claim that the lover enjoys divine protection at all times). The last epigrammatic theme discussed is that of the poet's attraction to several different types of girls/boys (Prop. 2.22A, 2.25.41ff; Tib.l.4.11ff; Ovid Am.2.4.2.10.). The fourth chapter is devoted to three illustrations of the elegist's obsequium his willingness to attend the girl in sickness, to hunt with her or go on a long journey with her. These, it is demonstrated by examples from Greek prose works on Friendship, are instances of the duties to be expected of a friend in Hellenistic Friendship Literature. These Freundschaftsdienste have been transformed by the elegists into examples of the lover's devotion. Chapter five is devoted to the themes of the girl's sickness (Prop. 2.28, (Tib.) 3.10, Ovid Am. 2.13), and her preoccupation with cosmetics (Prop.1.2, 2.l8c; Tib.l.8.9-16; Ovid Am.l.l4, Ars.3.101ff ., RA 343ff.). The former (which may derive ultimately from Callimachus) receives very different treatment from the three poets, though certain topoi recur in the poems, demonstrating a degree of inter-borrowing amongst the three. (The view that these similarities are due to the poems belonging to the genre soteria is countered in some detail). The treatment of the latter theme by Propertius may reveal comic influence, but its widespread occurrence in Greek literature of various genres precludes certainty. The conclusion attempts to draw together some of the threads of the discussion (which suggests throughout that while the elegists know and use Greek literature they are by no means servile imitators) by examining in detail Propertius 4.7 in which a number of different literary traditions are combined and used effectively by the poet.

A number of dihydroxyacetone kinases (DAK) have been described that can utilize either ATP (in animals, plants and some bacteria) or phosphoenolpyruvate (PEP) (in most bacteria) as the energy source to convert dihydroxyacetone (Dha) to dihydroxyacetone phosphate (DhaP), which plays critical roles in many metabolic pathways. It has also been described that Homo sapiens DAK is able to regulate the innate immune system via its interaction with Melanoma differentiation-associated protein 5 (MDA5), which is recognized as an RNA sensor during virus infection. These findings make H. sapiens DAK a very noteworthy research target due to its multiple functions in many fields. Therefore, structural studies of DAK from H. sapiens are presented. The initial structure of the wild type (WT) H. sapiens DAK was determined to 2.5Å resolution and solved by molecular replacement. The structure forms a homodimer and four dimers were shown to be present in each asymmetric unit. However, within each monomer, most regions of the C-terminal domain were disordered, and therefore in order to improve structure quality, multiple site-directed mutagenesis was used. The mutated protein was then crystallized and the structure was determined to 1.4Å. The N-terminal Dha binding domain consists of two α/β regions and the C-terminal ATP binding domain is comprised of eight anti-parallel α-helices, which forms a deep pocket and is filled with a phospholipid molecule. In addition, the structures of mutated DAK in complex with ATP analogues and Dha are also described in the current study. The second part of the project concerned sialidases, which are glycoside hydrolases that specifically hydrolyse terminal sialic acid from various glycans. Streptococcus pneumoniae is one of the most common pathogenic bacteria of humans, and is reported to encode three sialidases that act as virulence factors in bacterial colonization and infection. One of these sialidases, NanA, was reported to be present in all clinical strains and plays a vital role during the bacterial infection. Consequently, the structure of N-terminal Carbohydrate-binding module (CBM) domain of NanA has been determined to 1.8Å, and reveals a β-sandwich fold. The apo form of NanA-CBM is present as a dimer in the asymmetric unit, whereas a monomer was detected when it is bound to sialic acid or its derivatives. Structural comparisons between NanA-CBM and other structures of the CBM40 family were also performed. The substrate binding sites of NanA-CBM forms a cavity that is able to accommodate the substrates. A potential molecular binding site located beside the sialic acid binding site was revealed, and is occupied by the side chain of a lysine from a symmetry- related molecule. Heteronuclear single quantum coherence (HSQC) NMR spectroscopy and fluorescent-based thermal shift assays were also carried out to further characterise the protein. The current results reveal the structure of both DAK from H. sapiens and NanA-CBM from S. pneumoniae, which may contribute to a better understanding towards cell metabolism and bacterial colonization.

This study has been mainly focused on high temperature solid oxide electrolysis cells (HT-SOECs) for steam electrolysis. The compositions, microstructures and metal catalysts for SOEC cathodes based on (La₀.₇₅Sr₀.₂₅)₀.₉₅Mn₀.₅Cr₀.₅O₃ (LSCM) have been investigated. Hydrogen production amounts from SOECs with LSCM cathodes have been detected and current-to-hydrogen efficiencies have been calculated. The effect of humidity on electrochemical performances from SOECs with cathodes based on LSCM has also been studied. LSCM has been applied as the main composite in HT-SOEC cathodes in this study. Cells were measured at temperatures up to 920°C with 3%steam/Ar/4%H₂ or 3%steam/Ar supplied to the steam/hydrogen electrode. SOECs with LSCM cathodes presented better stability and electrochemical performances in both atmospheres compared to cells with traditional Ni cermet cathodes. By mixing materials with higher ionic conductivity such as YSZ(Y₂O₃-stabilized ZrO₂ ) and CGO(Ce₀.₉Gd₀.₁O₁.₉₅ ) into LSCM cathodes, the cell performances have been improved due to the enlarged triple phase boundary (TPB). Metal catalysts such as Pd, Fe, Rh, Ni have been impregnated to LSCM/CGO cathodes in order to improve cell performances. Cells were measured at 900°C using 3%steam/Ar/4%H₂ or 3%steam/Ar and AC impedance data and I-V curves were collected. The addition of metal catalysts has successfully improved electrochemical performances from cells with LSCM/CGO cathodes. Improving SOEC microstructures is an alternative to improve cell performances. Cells with thinner electrolytes and/or better electrode microstructures were fabricated using techniques such as cutting, polishing, tape casting, impregnation, co-pressing and screen printing. Thinner electrolytes gave reduced ohmic resistances, while better electrode microstructures were observed to facilitate electrode processes. Hydrogen production amounts under external potentials from SOECs with LSCM/CGO cathodes were detected by gas chromatograph and current-to-hydrogen efficiencies were calculated according to the law of conservation of charge. Current-to-hydrogen efficiencies from these cells at 900°C were up to 80% in 3%steam/Ar and were close to 100% in 3%steam/Ar/4%H₂. The effect of humidity on SOEC performances with LSCM/CGO cathodes has been studied by testing the cell in cathode atmospheres with different steam contents (3%, 10%, 20% and 50% steam). There was no large influence on cell performances when steam content was increased, indicating that steam diffusion to cathode was not the main limiting process.

The G-protein coupled receptors (GPCRs) comprise the largest family of receptors in humans and other vertebrates. They are embedded in the cell membrane and are activated by many different signaling molecules. Activation modulates cellular signal transduction pathways and influences many physiological processes. Therefore the GPCRs are important as targets for numerous drugs. The receptors for NPY (neuropeptide Y) belong to GPCRs of Class A (rhodopsin-like). NPY and its related peptides PYY and PP are involved in the regulation of appetite, blood pressure and many other processes. They share a common structure and interact with the receptors Y1, Y2, Y4 and Y5 in mammals, and, in addition, Y7 and Y8 in amphibians and bony fishes. This thesis is focused on the human Y2 receptor, known to reduce appetite, by investigating the importance of thirteen amino acid residues for ligand binding. Mutagenesis followed by functional expression and receptor binding was conducted. During the course of this work several new GPCR crystal structures have been resolved, thereby improving the receptor modeling in papers I-III. The major finding is that even though the Y1 and Y2 receptors have evolved from a common ancestor, their points of ligand interaction differ and have thus changed during evolution. In general, the positions investigated resulted in milder changes in the ligands’ affinities for Y2 compared to Y1. These findings were incorporated in the design of new Y1 and Y2 receptor models, leading to improved understanding of how such divergent receptors, sharing only 30 percent sequence identity, can still interact with the same ligands. Notably, several of the mutations introduced in Y2 resulted in increased affinity. A novel NPY receptor gene named Y2-2 was identified in the genomes of zebrafish and medaka. This brings the number of zebrafish NPY receptors to seven. The binding characteristics of zebrafish Y2-2 differed from zebrafish Y2 mainly in the interaction with NPY13-36 and the antagonist BIIE0246. In conclusion, these results increase our understanding of ligand interactions with GPCRs and will be useful for refinement of ligand-receptor models for future development of receptor subtype-selective drugs.

No description available.

This thesis describes a number of studies on organic semiconductors as laser gain media with the aim of simplifying the excitation scheme and exploring potential applications. A hybrid device taking the advantage of high power inorganic light emitting diodes (LEDs) and low threshold organic distributed feedback lasers is demonstrated to realize a LED pumped organic laser. When the drive current is higher than 152 A, a sharp peak is clearly observed in the laser output spectrum, implying the LED successfully pumps the polymer laser above threshold. This is the first time an incoherent LED has been used as the excitation source for an organic semiconductor laser. A strategy for further improving the performance of the hybrid device is explored with the use of a luminescent concentrator made of a dye doped SU8 film, to intensify the power density from the inorganic LED. The luminescent concentrator is capable of increasing the incident power density by a factor of 9 and reducing the lasing threshold density by 4.5 times. As a preliminary investigation towards mode-locked polymer lasers, the impact of a solid state saturable absorber on a solution based organic semiconductor laser is explored. The dye doped polystyrene thin film saturable absorber exhibits a saturation intensity of a few MW/cm². When it is placed into the laser cavity, a train of short pulses is generated and the underlying mechanism is discussed. Finally, the potential of using organic semiconductor lasers in the detection of nitro-aromatic explosive vapours is studied in distributed feedback polyfluorene lasers. A high sensing efficiency and fast response from the laser prove polyfluorene lasers can be used as disposal and low cost devices in explosive chemosensing.

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.