This work is focused on the topic of school class climate and its enhancement by the methods of personality and social education in a concrete class where the author is working as a teacher. The theoretical part is dedicated to the class climate and its characteristics. It describes what influences, by which methods it can be investigated and what can it be influenced by. In the practical part the author uses the methods of the teacher's active research to investigate the climate in her own class and also self-reflexively examines herself from the view of the beginning teacher. During the time period of one school year continually monitors the climate in her class and states the most effective ways towards improvement. The results of the research show that the methods of personality and social education have an influence to the class climate if they are used in a natural and casual manner. KEY WORDS: Class climate, teacher's active research, beginning teacher, social skills, self - reflection, begin together, personality and social education, cooperation, relations, pre - puberty, problematic pupil, method.

The aim of this thesis is to analyze a media campaign called Responsible Together, which is focused on decrease of violence against women. Campaign Responsible Together is created by non-profit organizations that are part of civil society. This thesis brings a description of the creative process of the campaign, it's target group, and communication mix. One part of this thesis also introduces the specifics of marketing in the non-profit field. A case study was selected as a research design because it allows combining more than one method of collecting data. These methods were document analysis, analysis of graphics, and participatory observation.

Text in Afrikaans / Die lied en musiekinstrumente speel 'n baie belangrike rol in die Bybel. Die Moseslied (Eks 15) word in die Psalms aangehaal en kulmineer in die 11 nuwe 11 lied van Openbaring - die lied van die "nuwe" hemel en die 11 nuwe 11 aarde wat verlostes saam sal sing. Die brandende vraag wat "reg" of hoegenaamd standpunt ingeneem moet perspektief (die Woord van God) "verkeerd" is en of daar word, word uit 'n Bybelse beantwoord, gegrond op die beginsel van die Reformasie: ecclessia reformata semper reformandum. Die geskiedenis van die lied van die vroee kerk tot by die Ref ormasie word kursories behandel. Die lied wat in die geskiedenis uit die mond van die gelowige, die lidmaat gehaal is en aan die priesters oorgelaat is, word deur Luther weer teruggeplaas in die mond van die gelowige. Dit blyk dat Calvyn, tsv die Ref ormasie slagspreuk, in sommige aspekte van sy beskouing van die kerklied, steeds 'n Roomse standpunt bly handhaaf en net tot by die vroee kerk "gereformeer" het. Slegs sgn "heilige" liedere, direk uit die Bybel, musiek sender instrumentale begeleiding, kon gebruik word. Saam met ons voorsate, wat van oa Nederland gekom het, het ons liederebundels gekom. Van Riebeeck het met die Datheense Psalter (die amptelike Nederlandse kerklied) wat later deur die 1805- "Evangelische Gezangen" aangevul is, geland. Agv die "cuius regio eius religio"-beginsel het die Hollandse kerklied voorkeur bo Lutherse ea kerkliedere gekry. Die 1805-"Evangelische Gezangen", alhoewel dit hartlik verwelkom is, het gelei tot misnoee in Colesberg en later in Rustenburg, wat op sy beurt gelei het tot kerkskeuring. Die "Evangelische Gezangen" was ook, naas die Bybel en die Psalmboek die metgesel van die Trekkers, kommandolede (Anglo-Boereoorlog) en die krygsgevangenes in die konsentrasie- en krygsgevangenekampe. In die kampe was daar baie tyd vir die sing en skep van gewyde liedere. Nie-goedgekeurde bundels en die vertaling van die Bybel in Afrikaans (1933) het aanleiding gegee dat liederebundels hersien moes word. Die 1937 (Totius-beryming), die 1944 Gesangboek, die 76/78-Psalmboek en die 1978 Gesangboek was die resultaat. 'n Dualistiese standpunt, dat sekere liedere geskik is vir kerkgebruik en ander slegs vir buite-kerkgebruik (die 1984- Jeugsangbundel) het aanleiding gegee tot die publikasie van SOM en Jeugsangbundel 2, 1993. Huidig gebruik talle gemeentes 'eie' bundels tsv sinodale besluite dat slegs SOM en JSB2 naas die Psalm- en Gesangboek gebruik mag word. Die verskyning van die Proefsangbundel 1998 (IKSA) lei 'n nuwe fase van die kerklied in SA in as die Ned Herv Kerk ook begin om "nuwe" liedere op die proef te stel. Vos, Strydom, Huisamen, Konig, Kloppers, ea het in resente jare evaluerings oor die kerklied gedoen. Die "ou", die "nuwe" en die kontemporere kerklied (veral met verwysing na die NG Kerk) m6et almal plek kry in die musiekskat van die 21ste eeu. / The hymn and musical instruments play a cardinal role in the Bible. The Song of Moses (Ex 15) is used in Psalms and in Revelation 15:3. The question with regards to what is acceptable and unacceptable and whether it is at all imperative to take a defendant stance, is clarified from a Biblical point of view. It is based on the Reformation-principle: ecclesia reformata semper reformandum. The History of the 'Song' Hymn in the early church up to the Reformation is shortly mentioned. The "songs" (Hymns) of the believer were in the course of history transferred to the priests and were only in the Reformation once again bestowed on the believer by Luther. It would appear that Calvin, despite the slogans of the Reformation continued to postulate a Catholic point of view with regard to the Hymns and that the Reformation was restricted to the early church. Only socalled holy songs, direct from the Bible and songs without instruments/accompaniment could be usedo The collection of 'songs' (Hymns) of the Dutch Reformed Church is an inheritance from the Netherlands. It was Van Riebeeck who brought in the ''Datheense Psalter'' . Later, the 1805 - "Evangelishe Gezangen" were added to it. Owing to the cuius regio eius religio-principle, the Dutch Church Hymn was given preverance. Although heartily welcomed, the "1805-Evangelische Gezangen" lead to a split in the Church. The "Evangelische Gezangen" (Hymns), and the Psalmbook were used by the 'Trekkers' , the 'Kommando' s' , (Anglo Boer war) and in the Consentration and prisoners of war camps. In the camps was ample time for singing and composing new sacred songs. 'Non-approved' song books and the translation of the Bible in Afrikaans (1933) gave rise to the birth of various Hymn and Psalter books. Youth Praise and Worship books were also published. The Dutch Reformed Church opinion that certain songs in the formal Church inappropriate and should gatherings. struggles with the dualistic are suitable for worshipping service while other are only be used at informal / Theology / D.Th. (Theology)

Der Dialog nach David Bohm darf nicht als bloße Kommunikationsmethode verstanden werden, er stellt ein eigenes, komplexes Paradigma dar. Daher verfolgt die Arbeit den Anspruch, das bohmsche Gedankenmodell in seiner Ganzheit zu veranschaulichen. Dieses Forschungsziel ist bisher einmalig. Es gibt keine wissenschaftliche Untersuchung, die die Genese des bohmschen Dialogs in seiner interdisziplinären Komplexität aufschlüsselt. Gleichzeitig existiert keine Forschungsarbeit, die den Dialog als didaktisches Medium versteht und überprüft. Die bisherigen Forschungsansätze haben den Dialog als Medium der Organisationskommunikation zur Ergebnisoptimierung wissenschaftlich erschlossen, sein pädagogisches Potential für andere Bereiche blieb dabei unbeachtet. Gerade als Medium der Schul- und Hochschuldidaktik ist er noch nicht wissenschaftlich ergründet worden. Diesem Anspruch stellt sich die Arbeit. Im Hinblick auf die Literaturdidaktik wird erforscht, in wie weit der Dialog nach David Bohm durch seine ontologische Erklärungsgrundlage, seine Prinzipien und Regeln eine kooperative Erkundungsmethode bietet, die das Spannungsfeld zwischen individuellen Deutungshorizonten und werkimmanenter Sinnstruktur offen legt. Dabei wird die Zielsetzung Bohms verfolgt und der Dialog als prozessorientiertes Kommunikationswerkzeug verstanden, dessen Zweck im gemeinsamen Denken und in der Ergründung der Schlussfolgerungs- und Denkprozesse liegt. Beim Blick auf die gegenwärtige formale Gestaltung des Dialogs zeigt sich, dass ein Einbezug psychologischer Phänomene bisher nicht ausreichend vollzogen ist. Die Arbeit schließt eine Forschungslücke, indem sie die Bedeutung des kooperativen Denkens und die dafür notwendigen Rahmenbedingungen erörtert, um eine Übertragung des Lernkonzeptes in die Literaturwissenschaft zu ermöglichen. So ist es das Ziel der Arbeit, den dialogischen Übungsraum durch ausgewählte didaktische Elemente zu ergänzen, die in besonderer Weise geeignet sind, Denkparadoxien kenntlich zu machen und dadurch eine greifbare Erklärungsgrundlage liefern, auf deren Basis eine dialogische Haltung ausgeformt werden kann. Erst die kritische Auseinandersetzung mit der metaphysischen Grundlegung und der methodischer Ausformung des Dialogs eröffnet die Gelegenheit das Kommunikationsmodell für andere Fachbereiche, beispielsweise Literaturdidaktik und Geschlechterforschung zu erschließen. Bei der Übertragung des bohmschen Konzeptes in die Literaturdidaktik fällt die große Kongruenz zwischen rezeptionsästhetischer Haltung und der bohmschen Ontologie auf. Dem Leser wird die Rolle eines aktiven Teilhabenden zugewiesen, der die Sinnsetzung durch Ausfüllen der literarischen Leerstellen entscheidend prägt. Seine Vorannahmen, Werte und Erfahrungen fießen in die Beurteilung der Figuren und die Deutung des Handlungsverlaufs ein, so dass sich eine individuelle Interpretation entwickelt. Die Vorstellungsbilder sind dabei keine wahllosen Muster, sie replizieren, umkreisen und nuancieren verschiedene Aspekte des zugrunde liegenden Werkes. Der eröffnende Kommunikationsstil des Dialogs ermöglicht es, den Geltungsbereich eigener Interpretationsweisen abzustecken, so dass die Komplexität und die Vieldeutigkeit als wesentliches Kennzeichen des Erkundungsprozesses hervorgehoben wird. Die Konzentration liegt nicht auf dem Abschließen und Beantworten, dem fnalen Lösen einer Frage, sondern auf der Entfaltung der Perspektivenvielfalt.:Inhaltsverzeichnis Einleitung: Dialogische Prinzipien in der Literaturwissenschaft........................................7 1 Die bohmsche Ontologie des Holomovements............................................................22 1.1 Vergleich der philosophischen Ableitungen aus Mechanik und Quantenphysik..........24 1.2 Die zwei Ordnungssysteme nach Bohm: Implicate und Explicate Order....................27 1.3 Die Überwindung traditioneller Dualismen..................................................................33 1.4 Bedeutungswandel tragender epistemischer Begriffe................................................39 1.5 Radikaler Konstruktivismus und Ontologie des Holomovements – ein Vergleich........43 1.6 Wissenschaftstheoretische Konsequenzen der pluralistisch-holistischen Weltordnung .........................................................................................................................................45 1.7 Die Ontologie des Holomovements als Chance für den Geschlechterdiskurs............50 2 Untersuchung der psychologischen Denkparadoxien.................................................61 2.1 Bedeutung der Holomovementontologie für den Bewusstseinsbegriff.......................61 2.2 Fragmentierung – Der Denkprozess als Wurzel der Inkohärenz..................................63 2.3 Systemischer Kreislauf des Denkprozesses - Gefühle, Gedanken, Muskelregungen..64 2.4 Einfuss mentaler Repräsentationen auf den Denkprozess.........................................69 2.5 Reaktionsweise des Denkens – refexhafter Abruf von Wissen...................................76 2.6 Konditionierung – Stabilität und Einfuss kultureller Annahmen...................................78 2.7 Selbstbildparadoxien – Abwehrmechanismen bei der Informationsverarbeitung........82 2.8 Die Anfälligkeit des Denkens für Fehlurteile und kognitive Illusionen...........................89 3 Die Methodik des bohmschen Dialogs..........................................................................93 3.1 Die Einfüsse des Philosophen Jiddu Krishnamurti auf das Dialogkonzept.................93 3.2 Die Einfüsse des Psychotherapeuten Patrick DeMare auf das Dialogsetting...........101 3.3 Defnition Dialog – eine Abgrenzung gegenüber der Diskussion...............................107 3.4 Schwierigkeiten im Dialogprozess............................................................................110 3.5 Gestaltungsregeln für das Dialogsetting...................................................................114 3.6 Die tragenden Prinzipien des Dialogprozesses – Propriozeption und Partizipation...118 3.7 Zusammenfassung: Dialogische Haltung durch dialogische Prinzipien?...................127 4 Der Dialog als pädagogisches Konzept......................................................................131 4.1 Vergleich dialogisch-pädagogischer Gruppengesprächssettings.............................132 4.2 Spezifsches Lernfeld des bohmschen Dialogs........................................................139 5 Neugestaltung der Dialogmethode..............................................................................146 5.1 Übungsanregungen zur Stärkung der gleichberechtigten Teilhabe...........................147 5.1.1 Praxisfeld Zuhören............................................................................................148 5.1.2 Praxisfeld Respektieren....................................................................................153 5.1.3 Praxisfeld Erkunden (Suspendieren).................................................................159 5.1.4 Praxisfeld Sprechen..........................................................................................165 5.2 Übungsanregungen zur Förderung refexiver Fähigkeiten.........................................167 5.2.1 Erkenntnisse der Action Science......................................................................169 5.2.2 Metakognitive Methoden des Kritischen Denkens............................................172 5 5.2.3 Umgang mit gesellschaftlichen Stereotypen anhand des Anti-Bias-Modells.....177 5.3 Techniken des kritischen Erkundens........................................................................178 5.3.1 Orientierungsfragen..........................................................................................179 5.3.2 Kriterien für die Beurteilung von Argumenten....................................................181 5.3.3 Frame Experiments..........................................................................................183 5.3.4 Hypothesentesten............................................................................................186 5.4 Weiterentwicklungspotentiale der Dialogmethode....................................................187 6 Das Anwendungsfeld des Dialogs in der Literaturwissenschaft...............................193 6.1 Herausforderungen einer geschlechtergerechten Pädagogik...................................193 6.2 Fazit und Kritik - Der Dialog nach Bohm als pädagogisches Medium......................198 6.3 Rezeptionsästhetische Gendersensibilisierung durch den Dialog.............................203 6.4 Zukünftige Forschungsfelder und Forschungsfragen – ein Ausblick.........................215 Danksagung......................................................................................................................220 Literaturverzeichnis...........................................................................................................221 / The ideal of communication developed by David Bohm offers a broad field of experience in discovering own judgements and assumptions through the skills of \"thinking together.\" It can be used in gender sensitive didactics. It is also a valuable tool for science discussions to get an open spectrum of perspectives regarding on one critical issue and to reflect together the relations between them.:Inhaltsverzeichnis Einleitung: Dialogische Prinzipien in der Literaturwissenschaft........................................7 1 Die bohmsche Ontologie des Holomovements............................................................22 1.1 Vergleich der philosophischen Ableitungen aus Mechanik und Quantenphysik..........24 1.2 Die zwei Ordnungssysteme nach Bohm: Implicate und Explicate Order....................27 1.3 Die Überwindung traditioneller Dualismen..................................................................33 1.4 Bedeutungswandel tragender epistemischer Begriffe................................................39 1.5 Radikaler Konstruktivismus und Ontologie des Holomovements – ein Vergleich........43 1.6 Wissenschaftstheoretische Konsequenzen der pluralistisch-holistischen Weltordnung .........................................................................................................................................45 1.7 Die Ontologie des Holomovements als Chance für den Geschlechterdiskurs............50 2 Untersuchung der psychologischen Denkparadoxien.................................................61 2.1 Bedeutung der Holomovementontologie für den Bewusstseinsbegriff.......................61 2.2 Fragmentierung – Der Denkprozess als Wurzel der Inkohärenz..................................63 2.3 Systemischer Kreislauf des Denkprozesses - Gefühle, Gedanken, Muskelregungen..64 2.4 Einfuss mentaler Repräsentationen auf den Denkprozess.........................................69 2.5 Reaktionsweise des Denkens – refexhafter Abruf von Wissen...................................76 2.6 Konditionierung – Stabilität und Einfuss kultureller Annahmen...................................78 2.7 Selbstbildparadoxien – Abwehrmechanismen bei der Informationsverarbeitung........82 2.8 Die Anfälligkeit des Denkens für Fehlurteile und kognitive Illusionen...........................89 3 Die Methodik des bohmschen Dialogs..........................................................................93 3.1 Die Einfüsse des Philosophen Jiddu Krishnamurti auf das Dialogkonzept.................93 3.2 Die Einfüsse des Psychotherapeuten Patrick DeMare auf das Dialogsetting...........101 3.3 Defnition Dialog – eine Abgrenzung gegenüber der Diskussion...............................107 3.4 Schwierigkeiten im Dialogprozess............................................................................110 3.5 Gestaltungsregeln für das Dialogsetting...................................................................114 3.6 Die tragenden Prinzipien des Dialogprozesses – Propriozeption und Partizipation...118 3.7 Zusammenfassung: Dialogische Haltung durch dialogische Prinzipien?...................127 4 Der Dialog als pädagogisches Konzept......................................................................131 4.1 Vergleich dialogisch-pädagogischer Gruppengesprächssettings.............................132 4.2 Spezifsches Lernfeld des bohmschen Dialogs........................................................139 5 Neugestaltung der Dialogmethode..............................................................................146 5.1 Übungsanregungen zur Stärkung der gleichberechtigten Teilhabe...........................147 5.1.1 Praxisfeld Zuhören............................................................................................148 5.1.2 Praxisfeld Respektieren....................................................................................153 5.1.3 Praxisfeld Erkunden (Suspendieren).................................................................159 5.1.4 Praxisfeld Sprechen..........................................................................................165 5.2 Übungsanregungen zur Förderung refexiver Fähigkeiten.........................................167 5.2.1 Erkenntnisse der Action Science......................................................................169 5.2.2 Metakognitive Methoden des Kritischen Denkens............................................172 5 5.2.3 Umgang mit gesellschaftlichen Stereotypen anhand des Anti-Bias-Modells.....177 5.3 Techniken des kritischen Erkundens........................................................................178 5.3.1 Orientierungsfragen..........................................................................................179 5.3.2 Kriterien für die Beurteilung von Argumenten....................................................181 5.3.3 Frame Experiments..........................................................................................183 5.3.4 Hypothesentesten............................................................................................186 5.4 Weiterentwicklungspotentiale der Dialogmethode....................................................187 6 Das Anwendungsfeld des Dialogs in der Literaturwissenschaft...............................193 6.1 Herausforderungen einer geschlechtergerechten Pädagogik...................................193 6.2 Fazit und Kritik - Der Dialog nach Bohm als pädagogisches Medium......................198 6.3 Rezeptionsästhetische Gendersensibilisierung durch den Dialog.............................203 6.4 Zukünftige Forschungsfelder und Forschungsfragen – ein Ausblick.........................215 Danksagung......................................................................................................................220 Literaturverzeichnis...........................................................................................................221

Text in Afrikaans / Die lied en musiekinstrumente speel 'n baie belangrike rol in die Bybel. Die Moseslied (Eks 15) word in die Psalms aangehaal en kulmineer in die 11 nuwe 11 lied van Openbaring - die lied van die "nuwe" hemel en die 11 nuwe 11 aarde wat verlostes saam sal sing. Die brandende vraag wat "reg" of hoegenaamd standpunt ingeneem moet perspektief (die Woord van God) "verkeerd" is en of daar word, word uit 'n Bybelse beantwoord, gegrond op die beginsel van die Reformasie: ecclessia reformata semper reformandum. Die geskiedenis van die lied van die vroee kerk tot by die Ref ormasie word kursories behandel. Die lied wat in die geskiedenis uit die mond van die gelowige, die lidmaat gehaal is en aan die priesters oorgelaat is, word deur Luther weer teruggeplaas in die mond van die gelowige. Dit blyk dat Calvyn, tsv die Ref ormasie slagspreuk, in sommige aspekte van sy beskouing van die kerklied, steeds 'n Roomse standpunt bly handhaaf en net tot by die vroee kerk "gereformeer" het. Slegs sgn "heilige" liedere, direk uit die Bybel, musiek sender instrumentale begeleiding, kon gebruik word. Saam met ons voorsate, wat van oa Nederland gekom het, het ons liederebundels gekom. Van Riebeeck het met die Datheense Psalter (die amptelike Nederlandse kerklied) wat later deur die 1805- "Evangelische Gezangen" aangevul is, geland. Agv die "cuius regio eius religio"-beginsel het die Hollandse kerklied voorkeur bo Lutherse ea kerkliedere gekry. Die 1805-"Evangelische Gezangen", alhoewel dit hartlik verwelkom is, het gelei tot misnoee in Colesberg en later in Rustenburg, wat op sy beurt gelei het tot kerkskeuring. Die "Evangelische Gezangen" was ook, naas die Bybel en die Psalmboek die metgesel van die Trekkers, kommandolede (Anglo-Boereoorlog) en die krygsgevangenes in die konsentrasie- en krygsgevangenekampe. In die kampe was daar baie tyd vir die sing en skep van gewyde liedere. Nie-goedgekeurde bundels en die vertaling van die Bybel in Afrikaans (1933) het aanleiding gegee dat liederebundels hersien moes word. Die 1937 (Totius-beryming), die 1944 Gesangboek, die 76/78-Psalmboek en die 1978 Gesangboek was die resultaat. 'n Dualistiese standpunt, dat sekere liedere geskik is vir kerkgebruik en ander slegs vir buite-kerkgebruik (die 1984- Jeugsangbundel) het aanleiding gegee tot die publikasie van SOM en Jeugsangbundel 2, 1993. Huidig gebruik talle gemeentes 'eie' bundels tsv sinodale besluite dat slegs SOM en JSB2 naas die Psalm- en Gesangboek gebruik mag word. Die verskyning van die Proefsangbundel 1998 (IKSA) lei 'n nuwe fase van die kerklied in SA in as die Ned Herv Kerk ook begin om "nuwe" liedere op die proef te stel. Vos, Strydom, Huisamen, Konig, Kloppers, ea het in resente jare evaluerings oor die kerklied gedoen. Die "ou", die "nuwe" en die kontemporere kerklied (veral met verwysing na die NG Kerk) m6et almal plek kry in die musiekskat van die 21ste eeu. / The hymn and musical instruments play a cardinal role in the Bible. The Song of Moses (Ex 15) is used in Psalms and in Revelation 15:3. The question with regards to what is acceptable and unacceptable and whether it is at all imperative to take a defendant stance, is clarified from a Biblical point of view. It is based on the Reformation-principle: ecclesia reformata semper reformandum. The History of the 'Song' Hymn in the early church up to the Reformation is shortly mentioned. The "songs" (Hymns) of the believer were in the course of history transferred to the priests and were only in the Reformation once again bestowed on the believer by Luther. It would appear that Calvin, despite the slogans of the Reformation continued to postulate a Catholic point of view with regard to the Hymns and that the Reformation was restricted to the early church. Only socalled holy songs, direct from the Bible and songs without instruments/accompaniment could be usedo The collection of 'songs' (Hymns) of the Dutch Reformed Church is an inheritance from the Netherlands. It was Van Riebeeck who brought in the ''Datheense Psalter'' . Later, the 1805 - "Evangelishe Gezangen" were added to it. Owing to the cuius regio eius religio-principle, the Dutch Church Hymn was given preverance. Although heartily welcomed, the "1805-Evangelische Gezangen" lead to a split in the Church. The "Evangelische Gezangen" (Hymns), and the Psalmbook were used by the 'Trekkers' , the 'Kommando' s' , (Anglo Boer war) and in the Consentration and prisoners of war camps. In the camps was ample time for singing and composing new sacred songs. 'Non-approved' song books and the translation of the Bible in Afrikaans (1933) gave rise to the birth of various Hymn and Psalter books. Youth Praise and Worship books were also published. The Dutch Reformed Church opinion that certain songs in the formal Church inappropriate and should gatherings. struggles with the dualistic are suitable for worshipping service while other are only be used at informal / Theology / D.Th. (Theology)

PWR, fuel assembly, benchmark, burnup, lattice, SCALE, Polaris, validation, reactivity

I mitt projekt utvecklar jag konstnärliga designmetoder för att studera, samla in och berätta om kvinnohistoria och interiörhistoria, inspirerat av feministiska och queerteoretiska perspektiv på temporalitet, identitet, historia, material och berättande. Mitt examensarbete utgår från min 90-åriga mormors berättelser om sina barndomsminnen av hennes två mormödrar ‘Momma’ och ‘Mormor’ och hur det var att spendera somrarna på deras gård, Fridhem. I formgivning av bordet Fridhem, tavlan Systrar på gungbräda i trädgården, installationen Flytande fragment och installationen av dessa under Konstfacks Vårutställning 2021 tillsammans med ljudverket Minnen från Momma och Mormors Fridhem, manifesterar jag studierna och berättelsen.

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.

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