The economic and population growth have led to the increased volumes of municipal solid waste in South Africa, hence more pressure to the waste management facilities. Municipal solid waste management is a by-product of everyday living, this is the reason why there is an environmental burden caused by waste to the environment. This study was conducted in Pretoria within the three sampled areas, namely: Garsfontein, Marabastad and Pretoria Central Business District (CBD). The rationale of the study was to establish the influence and effects of biographic factors (age, marital status, educational background, gender) and religious conviction on littering. This project constitutes both qualitative and quantitative data collection methods. About 150 of the questionnaires were administered to each of the three sampled areas. Therefore, the overall proportion of the respondents was 450. Visual inspection was done in all the three sampled areas to observe the littering status quo and waste management situation in these particular areas. The visual inspection uncovered very interesting findings. It was established that Marabastad was cleaner in the morning than in the late hours while Garsfontein was clean throughout the day. Another problem that was discovered, amongst others was that of the condition of the bins in Marabastad and Pretoria Central Business District (CBD) which was bad compared to Garsfontein. From the questionnaire, it was found that the 72% of elderly respondents (< 36 yrs) are more environmental conscious than the young people (18-35 yrs) who 83% of them claim to litter when there is no bin. Further, among young people, 78% said litter cigarette butts and 93% of these said better street cleanliness would help reduce littering. 72% of adults said religious convictions would help reduce littering. The chi-square and correlation coefficient statistical data analysis methods were used to establish the relationship between different biographic and religious variables. Gender, marital status, monthly income, age and religious conviction were all found to have significant effects on issues regarding littering in the city. / Environmental Sciences / M. Sc. (Environmental Science)

DNUR / Department of Advanced Nursing Science / See the attached abstract below

This study employed a qualitative approach to investigate the use of cooperative learning to enhance conceptual understanding of trigonometry in a Grade 11 mathematics classroom, conducted at a high school in Moletlane Circuit, Capricorn District in Limpopo Province, South Africa. A single case study was used as a research design to get an in-depth analysis and collect detailed data using semi-interviews and lesson observation of the cooperative learning of trigonometry in Grade 11 from the learners and the teacher. Participants were purposely chosen and consisted of (n=30) Grade 11 mathematics learners and their mathematics teacher. Data from the participants were collected through semi-structured interviews and observation, with the aid of observation guide (Appendix C) for three weeks. The salient findings from the study showed how cooperative learning was used, research questions, the approaches, the teacher did not adequately highlight the importance of trigonometry to students without integrating the topic to real-life situations. Some students said that the teacher did not teach trigonometry in a manner that they understood, which made trigonometry challenging for them. Concerning cooperative learning, the study found that many learners were passively engaged, listened to or watched the teacher. Mainly, the study recommends teacher-training institutions to host practical workshops to help teachers integrate theoretical training and practical cooperative learning experience. While this study was qualitative in nature, future researchers could conduct quantitative data collection. This would allow for the collection of numerical findings through survey questionnaires. / Mathematics Education / M. Ed. (Mathematics Education)

The subject of this master’s thesis is a solution of chosen parts of building construction project. The project solves industrial hall that contains productions and administrative space. This thesis includes technological regulations for substructure, study of performing the main technological stages, design of using machines, checking and testing plans. Other phrases parts are time schedules, budget, calculations, design of site equipment including engineering report and report for performing access road. Starting materials, which were used for this thesis, were engineering report also including working drawings.

Text in English with abstracts in English, Afrikaans and Venda / In this qualitative study, the research objective was to present a theoretical framework for the phenomenon of interpersonal leadership relations (denoting both the dyadic relationship between two leader/followers and the leadership communication taking place in the dyad) in knowledge-based organisational contexts. It is posited that the interpersonal leader-follower dyad (LFD) may be viewed from a systems theory perspective as a system consisting of two system parts (individuals). These individuals are labelled ‘leader/followers’ to emphasise their mutual interdependence, and to indicate that these roles may be interchangeable, based on the knowledge needs in a particular situation (in line with the tenets of shared leadership). The dyadic system is influenced by its environment, the organisational context. However, the primary focus of this study is on interpersonal leadership communication as symbolic interaction between the leader/followers in the LFD. These three systemic levels are represented as major themes in the model resulting from this study: Theme 1 – an organisational environment that supports constructive interpersonal leadership relations (ILR); Theme 2 – symbolic interaction in the LFD; and Theme 3 – personal attributes that enhance ILR. The data were collected from two convenience samples. In-depth, semi-structured interviews were conducted with participants in Sample 1, while questionnaires were used to collect data from Sample 2. In both cases, thematic analysis was used to analyse and interpret the data. The major contribution of the study is the resulting theoretical framework of ILR, which comprises a theoretically based definition of ILR; a generic model of ILR; and current guidelines for fostering constructive ILR in knowledge-based contexts, with reference to the three systemic levels. The following definition was phrased based on the study: Constructive interpersonal leadership relations (ILR) in a knowledge-based organisational context is a dyadic process of symbolic communication between two expert leader/followers who mutually influence each other and share meaning to strengthen their relationship and to collaboratively transfer and apply knowledge to achieve organisational goals. In terms of the environment, it was found that organisational leaders should actively model and promote the following: a collaborative leadership concept, workplace spirituality, cultural inclusivity, and adaptation to advancing communication technologies. Regarding symbolic interaction in the LFD, the following communication practices were found to be central to constructive ILR: active listening, supporting followers as unique individuals, respectful communication, considering followers’ input, facilitating constructive redefinition of the other leader/follower’s self, role-taking (taking the perspective of the other leader/follower’s role), awareness of attribution, conflict management through non-threatening, respectful and preferably face-to-face discussion, facilitating a sense of meaning or purpose at work for the other leader/follower, and fostering constructive relationship properties such as trust. It was found that ILR may produce system outputs into the organisation that contribute to the organisational culture and climate, job performance, employee morale and engagement, and staff retention. Personal attributes were organised into personal values and competencies that support ILR. The most important personal values were identified as honesty, love or supportiveness, respect, relationships or engagement, trust, and professional excellence. Essential competencies were identified as listening skills, emotional communication competencies (particularly self-awareness, self-reflection and attending to others’ emotions), engagement skills, conflict management skills, and multicultural competency (including generational skills). / In hierdie kwalitatiewe studie word ’n teoretiese raamwerk voorgelê vir die verskynsel ‘interpersoonlike leierskapsverhoudings‘ (verwysende na beide die diadiese verhouding tussen twee leier/volgelinge en die leierskapskommunikasie wat in die diade plaasvind) in kennisgebaseerde organisatoriese kontekste. Die uitgangspunt is dat die interpersoonlike leier-volgeling-diade (LVD) vanuit ‘n sisteemteoretiese perspektief beskou kan word as ‘n sisteem wat uit twee sisteemdele (individue) bestaan. Hierdie individue word ‘leier/volgelinge‘ genoem om hulle wedersydse interafhanklikheid te beklemtoon; en om aan te toon dat hierdie rolle uitruilbaar mag wees, afhangende van die kennisbehoeftes in ’n gegewe situasie (met verwysing na die teorie van gedeelde leierskap). As ’n sisteem word die LVD ook deur die omringende omgewing of organisatoriese konteks beïnvloed. Die primêre fokus van hierdie studie is egter op interpersoonlike leierskapskommunikasie as simboliese interaksie tussen die leier/volgelinge in die LVD. Hierdie drie sistemiese vlakke word in hierdie studie deur die hooftemas in die studie verteenwoordig en ook as sulks in die voortvloeiende model uitgebeeld: Tema 1 – ’n organisatoriese omgewing wat konstruktiewe interpersoonlike leierskapsverhoudings (ILV) ondersteun; Tema 2 – simboliese interaksie in die LVD; en Tema 3 – persoonlike eienskappe wat ILV bevorder. Die teoretiese raamwerk van ILV bestaan uit die volgende: ’n teoreties gefundeerde definisie van ILV; ’n generiese model van ILV; en ’n raamwerk van huidige riglyne vir die kweek van konstruktiewe ILV in kennisgebaseerde kontekste, met verwysing na die drie sistemiese vlakke van omgewing, diade en individuele leier/volgelinge. Die volgende definisie is op grond van die navorsingsresultate geformuleer: Konstruktiewe interpersoonlike leierskapsverhoudings (ILV) in ’n kennisgebaseerde organisatoriese konteks is ’n diadiese proses van simboliese kommunikasie tussen twee kundige leier/volgelinge wat mekaar wedersyds beïnvloed en betekenis deel om hulle verhouding te versterk en kennis samewerkend oor te dra en aan te wend om organisatoriese doelwitte te bereik. In terme van die organisatoriese omgewing is bevind dat organisatoriese leiers, veral senior leiers, die volgende aktief moet modelleer en bevorder in die organisasie: ’n samewerkende leierskapskonsep, spiritualiteit in die werkplek, kulturele insluiting, en aanpassing by vooruitgang in kommunikasietegnologie. Met verwysing na simboliese interaksie in die LVD is die volgende praktyke bevind as sentraal tot konstruktiewe ILV: aktiewe luistergedrag, die ondersteuning van volgelinge as unieke individue, respekvolle kommunikasie, die inagneming van volgelinge se insette, die fasilitering van die konstruktiewe herdefiniëring van die ander leier/volgeling se self, rol-inneming (die inneem van die rolperspektief van die ander leier/volgeling), bewustheid van attribusie, die bestuur van konflik deur nie-bedreigende, respekvolle en – waar moontlik – aangesig-tot-aangesig bespreking, die fasilitering van ’n sin van doel of betekenis by die werk vir die ander leier/volgeling, en die kweek van konstruktiewe verhoudingseienskappe (vertroue, uitruilbare leier/volgeling-rolle en wedersydse invloed is geïdentifiseer as belangrik). Dit is ook bevind dat ILV sisteemuitsette in die organisasie mag genereer wat bydra tot die organisatoriese kultuur en klimaat, werkprestasie, werknemers se moreel en betrokkenheid, en personeelbehoud. Persoonlike eienskappe is verdeel in waardes en vaardighede wat ILV ondersteun. Die belangrikste waardes is geïdentifiseer as eerlikheid, liefde, respek, verhoudings, vertroue, en professionele uitnemendheid. Die volgende vaardighede is geïdentifiseer as noodsaaklik: luistervaardighede, emosionele kommunikasievaardighede (met spesifieke verwysing na selfbewussyn, selfrefleksie en aandag aan ander se emosies), betrekkingsvaardighede, konflikbestuursvaardighede, en multi-kulturele vaardighede (wat generasievaardighede insluit). Die date is ingesamel uit twee gerieflikheidsteekproewe. Indiepte-, semi-gestruktureerde onderhoude is gevoer met Steekproef 1 (kundiges op gebiede verwant aan ILV in kennisgebaseerde kontekste), terwyl vraelyste gebruik is om data te verkry by Steekproef 2 (leier/volgelinge in kennisgebaseerde kontekste). Tematiese ontleding is in beide gevalle gebruik om die data te ontleed en te interpreteer. / Kha iyi ngudo ya u tandula ‘qualitative’, ndivho ya ṱhoḓisio yo vha u ṋetshedza mutheo wa thiyori kha sia ḽa vhushaka ha vhurangaphanḓa vhukati ha vhathu (zwine zwa amba vhushaka ha tshumisano vhukati ha vharangaphanḓa/vhatevheli vhavhili na vhudavhidzani kha vhurangaphanḓa vhune ha khou bvelela nga kha tshumisano yeneyo) kha nyimele ya tshiimiswa yo ḓitikaho nga nḓivho. Zwo sumbedziswa uri tshumisano ya murangaphanḓa-mutevheli vhukati ha vhathu (leader-follower dyad (LFD)) i nga lavheleswa u bva kha sia ḽa sisiṱeme ya thyori sa sisiṱeme ine ya vha na zwipiḓa zwivhili (vhathu). Vhathu avha vha vhidzwa ‘vharangaphanḓa/vhatevheli’ hu u itela u khwaṱhisedza u ḓitika havho nga muṅwe, na u sumbedza uri mishumo iyi i nga imelelana, zwo ḓitika nga ṱhoḓea dza nyimele yeneyo. (zwi tshi tevhedza vhatevheli vha vhurangaphanḓa uvho). Sisiṱeme ya tshumisano i ṱuṱuwedzwa nga nyimele yayo, nyimele ya tshiimiswa. Fhedziha zwa ndeme kha ngudo iyi ndi nga vhudavhidzani ha vhurangaphanḓa vhukati ha vhathu sa tshiga tsha tshumisano vhukati ha vharangaphanḓa/vhatevheli kha LFD. Maga aya mararu a sisisṱeme a imelelwa nga ṱhoho khulwane kha nḓila yo livhisaho kha ngudo iyi: Ṱhoho 1 – mupo/nyimele ya tshiimiswa i ṱuṱuwedzaho vhushaka ha vhurangaphanḓa vhu vhuedzaho vhukati ha vhathu (interpersonal leadership relations (ILR)); Ṱhoho 2 – Tshiga tsha tshumisano kha LFD; na Ṱhoho 3 – Vhuvha ha muthu vhune ha konisa ILR. Data yo kuvhanganywa u bva kha sambula dzine dza vha dza tsinisa. Mbudziso dzo ṱanḓavhuwaho, dzi sa langiho kufhindulele kha vhavhudziswa dzo itwa hu na vhadzheneli kha Sambula ya u thoma (1), ngeno khwesheya dzo shumiswa u kuvhanganya data kha Sambula 2. Kha nyimele dzoṱhe ho shumiswa ṱhaṱhuvho i re na vhushaka na ṱhoho u itela u ṱhaṱhuvha na u ṱalutshedza data. Zwine ngudo iyi ya vhuedza khazwo ndi mvelelo ya mutheo wa thyori wa ILR, ine ya vha na ṱhalutshedzo yo ḓitikaho nga thyori ya ILR, nḓila ya u angaredza ya ILR; na tsumbanḓila dza zwino u itela mbuelo ya ILR kha nyimele yo ḓitikaho nga nḓivho, zwo lavhelesa kha maga a sisiṱeme. Ṱhalutshedzo i tevhelaho yo vhekanywa zwi ḓitika nga ngudo: Vhushaka ha vhurangaphanḓa Vhuvhedzaho vhukati ha vhathu (ILR) kha nyimele ya tshiimiswa yo ḓitikaho nga nḓivho ndi maitele a tshumisano ya tshiga tsha vhudavhidzani vhukati ha vharangaphanḓa/vhatevheli vha re na nḓivho vhane vha ṱuṱuwedzana na u kovhekana zwine zwa amba u itela u khwaṱhisa vhushaka havho khathihi na u fhirisa na u shumisa nḓivho u itela u zwikelela zwipikwa zwa tshiimiswa. Zwi tshi ya nga nyimele, zwo wanala uri vharangaphanḓa vha tshiimiswa vha tea u vhumba na u ṱuṱuwedza zwi tevhelaho: muhumbulo wa tshumisano kha vhurangaphanḓa, zwa tshimuya mushumoni, u katela zwa mvelele, na u ṱanganedza u shumiswa ha thekhinoḽodzhi ya vhudavhidzani. Maelana na tshumisano nga tshiga kha LFD, maitele a vhudavhidzani a tevhelaho a wanala a one a ndeme kha ILR ire na mbuelo: u thetshelesa nga vhuronwane, u tikedza vhatevheli hu na kupfesesele kwa uri vhathu vho fhambana, vhudavhidzani ha ṱhonifho, u dzhiela nṱha mihumbulo ya vhatevheli, u ṱuṱuwedza u ṱhalutshedza nga nḓila yo fhambanaho i vhuedzaho ya vhaṅwe vharangaphanḓa/vhatevheli vha shumaho u ya nga vhone vhaṋe, u dzhia dzhenelela (u vhona nga nḓila ine vhaṅwe vharangaphanḓa/vhatevheli vha vona ngayo), u tangnedza nḓivho, ndaulo ya phambano nga kha nyambedzano i sa shushedziho, ya ṱhonifho, nga maanḓa nga nyambedzano vhathu vho livhana zwifhaṱuwo, u ṱuṱuwedza nḓivho ya ṱhalutshedzo kana ndivho ya mushumo kha vhaṅwe vharangaphanḓa/vhatevheli, u ṱuṱuwedza zwithu zwi fhaṱaho vhushaka vhu vhuedzaho u fana na u fulufhedzana. Zwo tumbulwa uri ILR i bveledza sisiṱeme ya mvelelo u vha tshiimiswa tshine tsha dzhenelela kha mvelele na vhuḓipfi, kushumele kwa mushumo, u ṱuṱuwedzea na u dzhenelela ha vhashumi, nauri vhashumi vha sa ṱuwe. U ṱanganedzea ha muthu zwo vheekanywa zwi tshi ya nga mikhwa ya muthu ene muṋe na vhukoni zwine zwa tikedza ILR. Mikhwa ya muthu ya ndemesa yo topolwa sa u fulufhedzea, lufuno, ṱhonifho kana u dzhenelela, fulufhelo, na vhukoni kha zwa phurofeshinaḽa. Vhukoni ha ndeme ho sumbedzwa sa vhukoni ha u thetshelesa, vhukoni ha vhudavhidzani ha muhumbulo (nga maanḓa u ḓiḓivha, u ḓilingulula/sedzulusa na u dzhiela nzhele vhuḓipfi ha vhaṅwe vhathu), vhukoni ha u dzhenelela, vhukoni ha ndaulo ya phambano, na vhukoni ha u dzhenelela kha mvelele nnzhi (zwi tshi katela vhukoni ha zwa murafho). / Communication Science / D. Litt. et Phil. (Communication)

This study addresses the concept of fair governance based on an empirical study with marginalized groups, primarily Muslims, and their interaction with government agencies as its salient locus of investigation. Employing the research method of in-depth interviewing, I present a qualitative analysis of 35 semi-structured interviews with Muslims and government administrators. The methodological framework based on which these interviews are interpreted is rooted in the tradition of social constructivism as manifested in the grounded theory perspective of Charmaz. My examination of the hitherto unspoken political visions of the study participants and their shared perspectives offers pragmatic solutions to create greater equity and fairer inclusion of the marginalized in civic and political dialogues and in the administrative practice of government. Remarkably, the cultural changes towards justice and inclusion in the Government of British Columbia manifest that fair government is committed to creating a fundamental transformation in favour of marginalized groups. I find the most promising approach for such transformation occurs where bottom up and dynamic approaches of civil society are aligned with top down approaches of government to justice. The findings suggest that fair governance enhances its functionality and capacity through reflecting universal universalism in its policies and practices, heartening public spirituality and moving towards a more humane modernity rather than the extant western model of modernity. Thus, fair governance calls for diversity in expression of religious identity and challenges the mistaken images of Muslim women. Subsequently, fair government welcomes female religious actors, who act upon religious values, to its administration and respects their choice of clothing encompassing the scarf. Fair government, at all levels, ameliorates the ethical standards of its employees and employs authentic leaders, who act in a virtuous manner, care about employees’ deeply held values, and implement direct communication with staff. Such government supports legislative and constitutional reforms to consider a different outlook of the marginalized on political and social concerns, respects religious practices, honours Muslims’ identity and interpretation of life, and supports individuals who aim to improve humanity in Canada and its occupational settings. Rethinking Islamophobia in the context of the distinct need of government administrators for the institutional education about Islam, as a key finding of the study, depicts the emergence of “Islamoexploria”, as a new expression, which I coin. In my study, there is ample evidence to suggest that a sample of government administrators in British Columbia is in the age of post Islamophobia since they, as pioneers, have passed the stage of Islamophobia and entered a new era of “Islamoexploria”. Thus, they have produced the profound socio-cultural changes towards understanding Islam by shifting from fear of, ostensibly, the unknown to knowledge about the unknown and to approaches that are more sympathetic to Muslims. This finding suggests that fair government facilitates the journey from western Islamophobia, a demonstration of old racism, to “Islamoexploria”, a contemporary thirst for knowledge about Islam. Concurrently, Muslims remain responsible to contribute to fairness at large by role modeling their religious values, which greatly promote justice, compassionate attitudes, and humanitarian actions. / Graduate / 2022-12-07

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.