Дисертації з теми "HR's ROLE"

Business is a very common and used words that stands for a variety of meanings. Todays concept of business is not just concerning in profit making. During the years managers and people in general has arisen a responsible commitment toward society. In this sense the concept of Corporate Social Responsibility has gaining in importance in the business context. During the years the literature review has framed different definitions, but the is still no standard approach on the topic. What is widely recognized is a linkage between CSR and Human Resource Management. Exactly the purpose of this dissertation is investigating which are the implications of CSR within HRM and vice versa. In doing this, a special attention is dedicated to sustainability issue. In order to better understand and look into this non simple relation some case studies will be introduced. After a theoretical review, a method will be design to provide a better picture of how, what and why the selected case studies respond to CSR policy evaluating the HRM policies. Appraising the impact that business have on society, an outlook will be useful to foster a long-term sustainable perspective. Key words: CSR; HRM; Sustainability.

This dissertation takes on a topic which is still somewhat unexplored and many of the originations have not paid heed to yet. Cyberbullying can be defined a “any use of information and communications technology to support deliberate and hostile attempts to hurt, upset or embarrass another person”. The technological advancement in the recent years has been imperative in changing the ways which business organizations work, it can be said that a modern-day organization will be rendered obsolete without the presence of technology. Technology is not only a choice of businesses but also has become a necessity to survive. In order to maximize the productivity and make an organization cost effective, inclusion of technology in almost every aspect of business is important. Recent developments in the work environment, specially the Covid-19 Pandemic has forced organizations to be heavily dependent on technology for every little thing. Increasing trend of remote working and cross-cultural hiring has also made technology and internet services a mandatory part of every organization. The virtues of technology, internet, and cyberspace as uncountable but a very big problem that is on the rise is the issue of cyberbullying in the workplace, it has been impacting the performances of the employees as well as their personal lives as well. Upon spending a considerable amount of time on researching literature, I concluded that the literature on Cyberbullying at workplace is very limited, though there has been a lot of work done on the area of workplace bullying as organizations are obviously concerned about their employees and do not want them to go through 6 something that is absurd in nature. The paucity of research in this area, especially in Indian context is what motivated me to choose this topic. This research work is carried out by doing extensive study on previous research papers or surveys conducted regarding Cyberbullying. The dissertation involves understanding the definition of the Cyberbullying and its context focusing on workplace environment. The act has been understood in reference to Indian organizations as well as workplaces abroad like UK, Canada, Australia, and Pakistan to understand the graveness of the act and how Governments and private organizations have formulated laws & policies to prevent as well to form a grievance redressal system for employees to approach for complaints without fear. The study also involves a primary component in which a survey is being carried out targeting working population in various private & Government organizations, and academic institutions to understand the sources of this act and if there is any counselling service provided to deal with same. Before embarking upon writing this dissertation, an initial survey revealed that it was women who majorly faced the wrath of cyberbullying and hence the main focus will remain on understanding this concern from women’s point of view and how workplaces have ensured that women don’t quit the job or remain stressed about these issues. The study further involves about examining various campaigns and initiatives taken by some organizations to make workplaces safer and cyberbullying free.

Världen står inför stora klimatutmaningar och det finns idag många verksamheter som har en negativ inverkan på miljö och klimat. Detta har lett till att FN har satt upp nya klimatmål som har inspirerat den svenska regeringen till en lagändring som medfört en ny hållbarhetslag i Sverige som syftar till att öka ansvarstagandet hos svenska företag. Syftet med den här studien var att undersöka hur HR-funktionen i några branscher på den svenska marknaden arbetar med miljömässig hållbarhet och arbetets övergripande frågor handlar om vilken roll HR har i miljömässigt hållbarhetsarbete samt hur de arbetar för att säkerställa kompetensen inom området. Arbetet har genomförts med en kvalitativ metod och fem semistrukturerade intervjuer. Av studien framkom att HR:s främsta roll i miljömässigt hållbarhetsarbete är som kommunikatör och avsändare samt att utbildning togs upp som en av de främsta bitarna där HR är med och bidrar till organisationens miljöprestationer.

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.

La première implication des récepteurs aux interférons de type 1 (IFNAR1) dans le contrôle de la voie Jak/STAT induite par les IFNs a été établie par mon laboratoire il y a une dizaine d'années (Marchetti et al., 2006). Une des questions fondamentales était alors de déterminer comment et pourquoi l'endocytose des IFNARs pouvait contrôler la voie Jak/STAT. Deux acteurs clés du tri endosomal ont attiré notre attention : Hrs (Hepatocyte growth factor-Regulated tyrosine kinase Substrate) et STAM (Signal Transducing Adaptor Molecule). Ces deux protéines constituent le complexe ESCRT-0 (Endosomal Sorting Complexes Required for Transport-0) les localisant, de manière idéale, à l'interface entre signalisation cellulaire et trafic membranaire.La combinaison de la biologie moléculaire et cellulaire, de la biochimie et de la microscopie à fluorescence, nous a permis d'établir que STAM s'associe au complexe IFNAR1 à la membrane plasmique afin d'y exercer son effet inhibiteur sur la signalisation Jak/STAT. Cette inhibition est levée lorsqu'IFNAR est libéré dans l'endosome et qu'il peut ainsi être recruté par Hrs sous la dépendance de l'IFN-α. En nous basant sur des expériences de déplétion par shRNA ou d'inhibition pharmacologique, nous avons identifié PTP1B (Protein Tyrosine Phosphatase 1B) en tant qu'activateur de la voie Jak/STAT induite par les IFNs. Le blocage sélectif de l'endocytose d'IFNAR par déplétion de clathrin, nous a permis de montrer que l'activation de PTP1B est inhibée à la membrane plasmique. Cela a été confirmé par des expériences d'interaction protéine-protéine (Proximity Ligation Assay) indiquant que STAM est constitutivement associé à IFNAR1, tandis que l'interaction entre IFNAR1 et Hrs a seulement lieu à l'endosome.Ainsi, nos résultats permettent d'établir un modèle dans lequel, à la membrane plasmique, STAM est un frein permanent à la signalisation Jak/STAT, qui est levé après l'endocytose d'IFNAR et sa libération dans l'endosome de tri. De plus, nous avons montré que l'interaction Hrs/STAM dans l'endosome précoce permet de différencier, de manière sélective, l'activation de la signalisation Jak/STAT médiée par l'IFN-α ou l'IFN-β
A decade ago, my laboratory established the first role of type I IFNs receptor (IFNAR) endocytosis in the control of Jak/STAT signaling induced by type 1 IFNs (Marchetti et al., 2006). A salient question is now to elucidate why and how IFNAR endocytosis could control the Jak/STAT pathway. Two key players of endosomal sorting retained our interest: Hrs (Hepatocyte growth factor-Regulated tyrosine kinase Substrate) and STAM (Signal Transducing Adaptor Molecule). These two classical components of the ESCRT-0 (Endosomal Sorting Complexes Required for Transport-0) complex were ideally placed at the interface between signaling and membrane trafficking. By using a combination of molecular and cellular biology, biochemistry, and fluorescent microscopy, we could establish that STAM binds to the IFNAR complex at the plasma membrane to exert an inhibitory effect on Jak/STAT signaling. This inhibition is removed when IFNAR is delivered to the sorting endosome by interacting with Hrs upon IFN-α stimulation. Based on shRNA down-expression and pharmacological inhibition, we further involve the PTP1B (Protein Tyrosine Phosphatase 1B) as it activates Jak/STAT signaling upon IFN stimulation. We could also show that PTP1B activation is inhibited by STAM at the plasma membrane from experiments where IFNAR endocytosis was blocked by siRNA-mediated clathrin down-expression. This was further confirmed by protein-protein interaction experiments (Proximity Ligation Assay) showing that STAM was constitutively associated with IFNAR1, whereas the interaction between IFNAR1 and Hrs occured only at the sorting endosome. Our results therefore allow to draw a model where STAM is a constitutive handbrake on Jak/STAT signaling at the plasma membrane that is released after IFNAR endocytosis and delivery to the sorting endosome. We further show that Hrs/STAM interaction at the early endosome allows to selectively distinguish the activation of Jak/STAT signaling mediated by IFN-α or IFN-β

‘Jardin de Granville’ ‘est une variété de rose moderne dédiée à des applications cosmétiques en lien avec ses propriétés intéressantes permettant de lutter contre les mécanismes inflammatoires et oxydants au niveau cutané. L’objectif principal de cette thèse a consisté à établir la cartographie moléculaire de ‘Jardin de Granville’. Pour cela, un procédé standardisé de récolte et d’extraction a été développé afin d’accéder au contenu moléculaire le plus exhaustif possible des différents organes de la plante. Des méthodes complémentaires d’analyse, allant de l’HPTLC, à l’HPLC-DAD-DEDL et jusqu’à l’UHPLC-HRMS, ont été mises en oeuvre pour réaliser les empreintes chromatographiques des extraits et en identifier les principaux constituants. Ces méthodes ont été choisies de plus en plus spécifiques et précises, de façon à apporter une graduation dans le niveau d’informations apportées. Plus de 120 molécules ont pu être caractérisées dans les différents extraits. Le deuxième objectif résidait dans la mise en évidence des marqueurs phytochimiques spécifiques à la variété en comparant ses empreintes moléculaires à celles des deux variétés parents. Deux méthodes de comparaison des profils ont été développées. La première met en jeu des analyses statistiques telles que l’ACP, la CAH et l’ANOVA qui permettent de comparer l’ensemble des extraits. La seconde effectue la soustraction des chromatogrammes d’extrait deux à deux et donne accès à un niveau d’informations plus ciblé. Ces deux approches ont conduit à l’identification de composés différenciant chaque type d’organes ce qui pourra servir d’outils dans la valorisation de certaines parties de la plante. Des marqueurs potentiels plus spécifiques à ‘Jardin de Granville’ ont pu être mis en évidence ce qui démontre la capacité des méthodes développées à différencier le contenu phytochimique de variétés de rose très proches
The modern rose variety ‘Jardin de Granville’, possesses proven activities against skin cell inflammatory and oxidant mechanisms and is devoted to cosmetic applications. The main goal of this study was to establish the molecular fingerprint of the different organs of ‘Jardin de Granville’. In this way, a standardized process for plant harvesting and sample extraction was developed giving access to the most exhaustive molecular fingerprint possible of the different organs. Several complementary analytical methods were implemented through HPTLC, HPLC-DAD-ELSD and UHPLC-HRMS, enabling to achieve the chromatographic fingerprint of the different organs and to identify the main constituents. These methods were selected to have increasing specificity and accuracy to bring progressive information on the molecule structure. Thus, more than 120 compounds were characterized in the different extracts. The second objective consisted in identifying specific phytochemical markers of the variety by comparing its fingerprint to those obtained from its two rose plant parents. In this way, two approaches were developed. The first one involves statistical analysis like PCA, HAC and ANOVA and allows comparing the whole sample chromatograms. The second approach performs extract chromatogram subtractions two by two and gives more detailed information. Both comparative methods led to the identification of the differential compounds existing between the different organ types which could be used to valuate some plant parts in particular. Some ‘Jardin de Granville’ specific markers were highlighted showing the method capacity to distinguish very close rose varieties, by comparing their molecular content

Today many organisations are transforming from a traditional structure to an agile structure to be able to work more efficiently, meet the fast changes that are continuously happening and make sure not be outperformed by competitors. Agile was from the beginning a set of values and principles designed for software development but it has scaled and today it includes the whole organisation. To avoid imbalance within the organization ,it is crucial that the whole organisation applies an agile mindset. However, the transformation may imply a lot of obstacles and challenges on the way. For some departments within an organisation, it is a lot easier to keep up with the changes and be a part of the transformation process while other departments lag behind or are forgotten, which is common for the HR department. HR ́s traditional responsibilities are performance appraisal, learning and development, compensation and recruiting and they are commonly viewed as an administrative function rather than a strategic function. However, it is important for HR to be a part of the agile transformation to be able to support the business. Applying agile can be a challenging task for HR since the requirements are many and the goal is clear which makes it hard to have an agile approach. On the other hand, HR can apply “agile lite” to their responsibilities and in that way fit the new organisation structure. In an agile transformation process it is highlighted that HR could function as a change agent, since they have a direct impact on the effectiveness of an organisation’s change and development. However, HR usually takes on a reactive role and are not the ones leading the transformation. This thesis examines two aspects for HR in an agile transformation process. First of all, the roles of HR in an agile transformation is investigated followed by identification of the challenges arising for HR during a transformation. How these challenges impact each other and the role of HR is also covered in the thesis. The study is based on qualitative research methods combined with a literature review. For the data collection, a total of seven semi-structured interviews with people from selected organisations were made. Further, a focus group session was conducted to discuss the findings and get a deeper understanding of the topic. To be able to analyze the aspects, theories concerning traditional HR, agile, agile applied to HR and the change process are covered in the thesis. Further, the chapter Cases of HR in agile transformations is categorized under HR’s role in an agile transformation and challenges with HR in an agile transformation. Regarding HR ́s role in an agile transformation there are different opinions. Concluded from this research, HR could act as a change agent. Further, what affects what role HR ends up with is mainly based on their reputation and if they understand and work close to the organisation, which is two identified challenges. Other main challenges identified for HR in an agile transformation are: Understand and work agile, HR’s traditional responsibilities, Experiment, Competence, Attitude and awareness of change, Understand the “why” and communicate continuously, Time and capacity, Behavioral knowledge and External factors.

Intresset för att studera intern kommunikation startade när vi själva insåg områdets komplexitet och utmaningar. Fascinationen över att kommunikation, som är ständigt förekommande i alla typer av sammanhang, kan vara så svårt att hantera bidrog till utformandet av studiens syfte och frågeställningar. Studien syftar till att undersöka hur en myndighet använder HR och den kompetens som HR besitter samt huruvida den interna kommunikationen fungerar som en integrerad funktion mellan ledning och chefer. Studien utgår från en kvalitativ ansats där fokus riktas mot respondenternas egna upplevelser och tolkningar.Studiens empiriska material är insamlat på en statlig myndighet genom fyra semistrukturerade intervjuer samt en minifokusgruppsintervju med tre medarbetare. Respondenterna representeras av chefer och medarbetare från myndighetens kommunikationsavdelning samt HR-funktion. Resultatet visar att intern kommunikation definieras som något som sker inom den egna organisationen och beskrivs som både det viktigaste och samtidigt det svåraste att förhålla sig till och hantera. Vidare visar studien att en önskvärd intern kommunikation präglas av ett öppet klimat, möjlighet till medarbetarinflytande samt ett mer uttalat samarbete mellan myndighetens HR- och kommunikationsavdelning. Tidigare forskning har framförallt fokuserats till intern kommunikation vid förändring, något som även inkluderats i studien då den undersökta myndigheten genomgår en övergripande organisationsförändring där samverkan mellan stödfunktionerna kommer öka. Resultatet påvisar att en av de utmaningar som intern kommunikation bidrar till utgörs av att skapa en gemensam förståelse för situationer och problem då olika befattningar och roller utgår från differentierade perspektiv.Nyckelord:

Bakgrund: Distansarbete till följd av den pågående Covid-19 pandemin har blivit ett hett ämne senaste året. HR har som alla andra yrkesgrupper påverkats av detta, HR är ett komplext yrke som kräver mycket mänsklig interaktion och därför kanske kan vara svårt att kombinera med ett distansarbete i alla lägen. Syfte: Syftet är att tolka om HR-rollen har förändrats inom organisationer i kontexten det ökade distansarbetet till följd av Covid-19. Metod: Denna studie har genomförts med hjälp av en kvalitativ datainsamling i form av semistrukturerade intervjuer och ett tolkande förhållningssätt. Datainsamlingen har sedan lett till en empirisk dataanalys som identifierade fyra teman i form av initialt krisarbete, det digitala arbetssättet, kommunikation i digitala sammanhang och framtiden efter covid-19. Slutsats: HR har behövt arbeta mer operativt än tidigare för att kunna hantera den problematik som Covid-19 har medfört. Detta har medfört att fokus skiftats ifrån de strategiska roller som finns inom HR. Därför har HR-rollen påverkats men i grunden inte förändrats.
Background: Teleworking as a result of the ongoing Covid-19 pandemic has become a hot topic in recent years. HR, like all other professions, has been affected by this, HR is a complex profession that requires a lot of human interaction and therefore may be difficult to combine with a telework in all situations. Purpose: The aim is to interpret if there is a change in the HR role within organizations in the context of the increased teleworking as a result of Covid-19. Method: This study was conducted using a qualitative data collection in the form of semi-structured interviews and an interpretive approach. The data collection has since led to an empirical data analysis that identified four themes in the form of initial crisis work, the digital approach, communication in digital contexts and the future after covid-19. Conclusion: HR has had to work more operationally than before to be able to handle the problems that Covid-19 has caused. This has meant that the focus has shifted from the strategic roles that exist within HR. Therefore, the HR role has been affected but has not fundamentally changed.

Inledning: Omorganiseringstrenden har varit stark i Sverige under 2000-talet. Ett av motiven har varit att HR-funktionens strategiska personalarbete kan bidra till kostnadseffektivitet och värdeskapande som behövs i en konkurrenskraftig miljö. Kunskapsintensiva organisationer i tillväxt är helt beroende av sitt humankapital och bör definiera behoven att ha ett utstuderat sätt att arbeta med HR.     Syfte: Syftet med studien är att förstå de behov och förväntningar som föreligger att implementera en formaliserad HR-funktion för kunskapsintensiva företag i tillväxt. Vi vill genom studien undersöka HR-funktionens roll och betydelse samt nyansera bilden av organisationens drivkrafter till att implementera en formaliserad HR-funktion.   Metod: Uppsatsen består av en induktiv ansats med en kvalitativ metod. Vi har genomfört en fallstudie på ett medelstort företag i tillväxt i en kunskapsintensiv bransch. All empirisk data har insamlats genom kvalitativa intervjuer på ledningsgruppnivå samt en enkät till samtliga medarbetare på fallföretaget.     Slutsats: De slutsatser vi kommit fram till är att behovet av att implementera en HR-funktion inom kunskapsintensiva företag i tillväxt beror på medarbetarnas attityder, motivation och beteende, som i sin tur påverkar organisationens resultat. De mjuka värdena bör gå hand i hand med de hårda värdena. Humankapitalet är en av de största konkurrensfördelar som morgondagens organisationer kommer att stå inför där tyngdpunkten ligger i att arbeta systematiskt med HR samt att transformera mål och strategier till att bli mer effektiva, värdeskapande och strategiska.
Introduction: Reorganization trend has been strong in Sweden in the 2000s. One of the reasons has been that the HR function's strategic in HR-work can contribute to cost efficiency and creates value, well needed in a competitive environment. Knowledge-intensive organizations in growth are entirely dependent on its human capital, and should examine the need to maintain a studied approach to working with HR.   Purpose: The purpose of this study is to understand the needs and expectations that are in place to implement a formalized HR function for knowledge-intensive companies in growth. We want to study the HR function's role and importance and nuanced the picture of the organization's incentives to implement a formalized HR function.   Method: The paper consists of an inductive approach with a qualitative method. We conducted a case study of a medium sized company in growth in a knowledge-intensive industry. We have collected all the empirical data through qualitative interviews in the management-team and a questionnaire/survey to all employees in the company.   Conclusion: The conclusion we reached is that the need to implement an HR function in knowledge- intensive companies in growth is context, expertise, size and leadership. Human capital is one of the major competitive advantages that tomorrow's organizations will face and the focus is on working systematically with HR and transform goals and strategies to become more efficient, value creation and strategic.

Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease. HTLV-1 Related Endogenous Sequence (HRES-1), a human endogenous retrovirus, produces 2 retroviral-like Gag capsid proteins (p8 and p15) that share significant sequence homology to the U1-subunit of the small ribonucleoprotein complex (U1sn-RNP), an autoantigen of lupus. The central hypothesis is that molecular mimicry between HRES-1 and U1sn-RNP serves as a priming event in SLE via the production of cross-reactive autoantibodies. Anti-HRES-1/U1sn-RNP serological responses in subjects with SLE and comparison populations were characterized. An overlapping peptide set mapping the HRES-1 p8 and p15 proteins was used. SLE subjects produce IgG to several regions of HRES-1. Healthy subjects or those with RA, HIV-1 infection, or HTLV-1-infection produced no significant anti-HRES-1 IgG. Anti-HRES-1 antibodies deposited in the kidneys of patients with SLE glomerulonephritis were identified. Our data suggests that HRES-1 plays a role in SLE by means of a molecular mimicry mechanism with U1sn-RNP.

Problemformulering: Vad har digitaliseringen inneburit för HR-avdelningar inom den offentliga sektorn? Syfte: Uppsatsens syfte är att identifiera hur digitaliseringen har påverkat HR-avdelningars arbete och roll, för att skapa en förståelse för vilka följder digitaliseringen har lett till för HR-avdelningarna. Metod: En induktiv ansats och kvalitativ datainsamling där det empiriska materialet samlades in med hjälp av kvalitativa intervjuer på informanternas arbetsplatser. Slutsats: Studien påvisar att digitaliseringen har inneburit och innebär en förändring för HR-avdelningar. Digitaliseringen har lett till att digitala verktyg introducerats som har påverkat många arbetsprocesser, och framför allt det administrativa arbetet. Digitaliseringen har även lett till konsekvenser för hur arbetet ser ut och hur bland annat kommunikation, relationer och effektivitet uppfattas. HRs roll har förändrats till att ha blivit mer stödjande, mer strategisk och antagit rollen till att vara en förändringsagent.

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.

Thesis (Ph. D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2006.
Title from PDF t.p. (viewed on Nov. 17, 2008) Includes bibliographical references. Also issued in print.