Das Ziel der vorliegenden Arbeit bestand darin, ein Vektorsystem zu entwickeln, dass den simultanen Transfer verschiedener Transgene in CD8+ und CD4+ T-Zellen und dadurch die Herstellung eines immunotherapeutischen T-Zell-Produkts ermöglicht, welches aus zwei unterschiedlich modifizierten T-Zell-Subtypen besteht. Im ersten Teil der Arbeit wurde die Targeting-Technologie von lentiviralen auf γ-retrovirale Vektoren übertragen. Anschließend wird die Herstellung von Vektoren beschrieben, die spezifisch für murines CD4 oder CD8 sind. Deren Spezifität wurde zum einen durch die exklusive Expression von GFP in CD4+ oder CD8+ Zellen und zum anderen durch den Dosis-abhängigen Verlust des GFP-Signals nach Inkubation dieser Zellen mit CD4- und CD8-blockierenden Antikörpern nachgewiesen. Im dritten Teil der Arbeit wird gezeigt, dass MVm8 und MVm4 primäre T-Zellen spezifisch transduzieren. MVm8-vermittelter Transfer des Ovalbumin (OVA)-reaktiven TZRs OT-I führte zu T-Zellen, die OVA+ Tumor-Zelllinien erkannten und Interferon-γ sezernierten. Der vierte Teil dieser Arbeit beschäftigt sich mit der in vivo Transduktion primärer T-Zellen mithilfe von MVm8, welches den OT-I-TZR und eine Luciferase transferiert (MVm8/OT-I-luc). Durch systemische Applikation von MVm8/OT-I-luc wurden T-Zellen in vivo transduziert. Durch Immunisierungen konnten antigen-spezifisches Homing, Expansion und eine anschließende Kontraktion in vivo transduzierter T-Zellen gezeigt werden. Mäuse mit starker OT-I-luc-Expression waren gegenüber einer Infektion durch OVA-transgene listeria monocytogenes geschützt. Zusammenfassend lässt sich sagen, dass das in dieser Arbeit entwickelte Vektorsystem in der Lage ist zwischen Subtypen von T-Zellen zu unterscheiden und sie simultan mit unterschiedlichen Transgenen auszustatten. Für MVm8 konnte gezeigt werden, dass es T-Zellen direkt in vivo transduzieren kann. / The aim of this thesis was to generate a vector system that allows the simultaneous transfer of different transgenes into CD8+ and CD4+ T cells, allowing the generation of a immunotherapeutic T cell product comprised of two differently engineered T cell subsets. The first part of the thesis describes the transfer of the measles virus (MV) envelope-based targeting technology from lentiviral (LV) to γ-retroviral (gRV) vectors. The second part reports the generation of two targeting vectors specific for murine CD4 or CD8. The exclusive specificity of MVm4 and MVm8 was proven by expression of GFP in CD4+ and CD8+ reporter cells, respectively, but not in CD4-CD8- cells after transduction, and by a dose-dependent loss of GFP signal after incubation of reporter cells with CD4 or CD8 blocking antibodies before transduction. The third part shows that MVm8 but not MVm4 transduced primary T cells. MVm8-mediated transfer of the ovalbumin (OVA)-reactive TCR OT-I resulted in T cells secreting interferon-γ (IFNγ) upon recognition of OVA+ tumor cell lines. The final part of this thesis describes the in vivo transduction of primary T cells using MVm8 transferring OT-I and a luciferase (MVm8/OT-I-luc). To this end, B6 mice deficient for Rag2 have been repopulated with either polyclonal (B6) or monoclonal T cells derived from P14-TCR transgenic mice (P14). One day later the transferred T cells were transduced in vivo by systemic application of MVm8/OT-I-luc. Upon immunization in vivo-transduced T cells homed, expanded and contracted repeatedly in an antigen-dependent manner. Finally, mice exhibiting strong luc-signals showed improved protection against infections by OVA-transgenic listeria monocytogenes (LM-OVA). In conclusion, the viral vector system developed within this thesis is able to discriminate between the two main T cell subsets and to equip them with distinct transgenes simultaneously.

The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible. / Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą]

Baigiamojo darbo tikslai – nustatyti neefektyvios žmogaus virusų glikobaltymų raiškos mielėse priežastis ir sukurti mielių kamienus su integruotais žmogaus ląstelių sekrecinio kelio genais bei ištirti jų įtaką glikobaltymų sintezei ir brendimui mielėse. Darbo eigoje pirmą kartą buvo aprašytos tymų viruso hemagliutinino (TVH) sintezės galimybės mielėse Saccharomyces cerevisiae ir Pichia pastoris. Parodyta, kad mielių ko-transliacinio baltymų perkėlimo į endoplazminį tinklą (ET) ir ET baltymų sulankstymo mechanizmai nėra pritaikyti sudėtingų virusinių baltymų brendimui, todėl klasikinės mielių rūšys ir standartiniai rekombinantinių baltymų raiškos ir gryninimo protokolai nėra tinkami diagnostikai ir vakcinų kūrimui reikalingo TVH baltymo gavimui. Proteominė S. cerevisiae ląstelių, sintetinančių TVH baltymą, analizė leido nustatyti kad, TVH sintezė mielėse sukelia neseniai literatūroje aprašytą citoplazminį nesusivyniojusių baltymų atsaką (UPR-cyto). Pagrindinis šiame darbe aprašyto atsako į stresą požymis yra ypatingai didelių baltymų agregatų, kurių šerdį sudaro TVH ir mielių eEF1A baltymai, susidarymas. Žmogaus tipo ko-transliacinį baltymų pernešimą į ET mielių ląstelėse bandyta atkurti perkeliant žmogaus SRP, Sec61 kompleksų ir TRAM1 baltymus, o siekiant sukurti tinkamas TVH baltymo brendimui sąlygas, mielių ląstelių ET buvo sintetinami pagrindiniai žmogaus ląstelių ET šaperonai – BiP, kalretikulinas, kalneksinas, PDI ir ERp57. Nors šiame darbe nepavyko sukurti mielių... [toliau žr. visą tekstą] / The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible.

This essay examines tweets published during a measles outbreak in January 2018 with the purpose of highlighting the division among tweets positive and negative toward vaccine, the use of sources and arguments as well as what risks are expressed and how the tweets relate to research and media. The study conducted is based on a combination of a quantitative content analysis followed by a qualitative content analysis. In total 260 Swedish tweets are being analyzed during the study, 18 of them make up the material for the qualitative study, all published 2-5 January 2018. Theories about media and health, post-truth, risk and participation make up the theoretical framework for the conducted study. The results from the study are analyzed through these theories. Results from the quantitative content analysis show that more than half of the tweets mentioning vaccine are positive toward vaccine. Tweets arguing in favor of vaccine use more sentiment in their arguments and those opposing vaccine more often use rational arguments. The quantitative analysis also show that half of the tweets used a source and primarily with the purpose of spreading information. Additionally a majority of those tweets using a source support the source mentioned, sources mostly used are media or authorities/institutions. Almost two-thirds of all tweets express some kind of risk. The risks most frequently expressed are risk of disease, lack of people vaccinated and immigration. Four themes emerge from the qualitative content analysis, two themes among tweets negative toward vaccine and two themes from tweets positive toward vaccine. The negative tweets speak of measles as a harmless disease and the danger of taking vaccine. On the contrary, the positive tweets speak of the dangers of measles and of the lack of people vaccinated as dangerous. / I denna studie undersöks twitterinlägg som publicerats under utbrott av mässling i januari 2018 för att belysa fördelningen mellan inlägg för och mot vaccin, användning av källor och argument samt hur inläggen ger uttryck för risk och även deras förhållande till forskning och media. Studien bygger på en kombinerad metod med en kvantitativ innehållsanalys följt av en kvalitativ innehållsanalys. Totalt har 260 svenska twitterinlägg ingått i studiens empiriska material, varav 18 ingått i den kvalitativa delen av studien, och samtliga är hämtade 2-5 januari 2018. Teorier om hälsa och medier, post-truth, risk samt deltagande utgör den genomförda studiens teoretiska ramverk. Dessa teorier används för att analysera studiens resultat. Resultatet av den kvantitativa analysen visade att mer än hälften av de inlägg som berörde vaccin var positiva till vaccin. I sin argumentation användes främst emotionella argument hos inlägg positiva till vaccin och logiska argument hos inlägg negativa till vaccin. Den kvantitativa studien visade även att hälften av alla inlägg använt sig av en källa och då främst i syfte att informera, dessutom ställde majoriteten av twitterinläggen sig positiva till den källa de hänvisat till vilket oftast var media eller myndigheter/institutioner. Nästan två tredjedelar av inläggen gav uttryck för risk och då var sjukdom, brist på vaccinerade och invandring de risker som lyftes fram mest. I den kvalitativa delen av studien framkom fyra teman, två för inlägg negativa till vaccin och två för inlägg positiva till vaccin. Inlägg negativa till vaccin talade om hur mässling inte var farligt och att vaccin var farligt. I motsats till detta talade inlägg positiva till vaccin om att mässling var farligt och att brist på vaccinerade var farligt.

Fatores associados com falhas na cobertura da vacinação contra o sarampo na cidade de São Paulo foram estudados por meio de entrevistas com pais ou responsáveis por 122 crianças de até cinco anos. Observou-se que a população conhece a doença e identifica a vacina com a idéia de proteção, mas não cumpre o calendário de vacinação e atrasa as doses da vacina contra o sarampo. Idade, região de origem ou de residência, status familiar ou nível educacional não mostraram associação com o cumprimento do calendário obrigatório (uma dose da vacina contra o sarampo deve ser aplicada dos 9 aos 11 meses e a segunda, aos 15 meses). A televisão é o meio de comunicação que mais informa os entrevistados sobre as Campanhas Nacionais de Multivacinação e o cartão de vacinação foi identificado como uma das vias de comunicação utilizadas para informar sobre a vacinação fora das Campanhas, na rotina. No entanto, nenhum meio de comunicação identificado no estudo pôde, significativamente, promover a mudança de comportamento da indife rença para o compromisso com o cumprimento do calendário, ou seja, adesão. A única variável significativamente relacionada com o atraso na vacinação, de até 20 dias, foi o sentimento de dó de aplicar injeções nas crianças (p = 0,08). Crianças brasileiras têm de visitar o posto de saúde para receber, até cinco anos de idade, 19 vacinas, 12 até o primeiro ano. O estudo deixa claro que não há adesão ao calendário proposto para vacinação contra o sarampo, apesar de ser esta a proposição das Campanhas Nacionais de Multivacinação e da vacinação de rotina / Risk factors associated with failure to receive measles vaccine were studied in eight districts of São Paulo city, Brazil. Parents or carers of 122 children were interviewed about their perception and understanding about the disease anda about measles vaccination, a kind of knowledge that can be acquired through vaccination communication campaigns or from personal communication (from the doctors, nurses of the health care center or friends). Population description factors were also identified. Results analysis shows that neither age, region of origin or residence, marital status nor educational level were related to taking or not taking measles vaccines adequately. Most of the people remembered about having being informed about the last annual vaccination campaign by television, but no communication channel was significantly associated with vaccination status. Answers to questions about knowing the disease or not or knowing the vaccine or not, when analysed alone, did not show any association with taking measles vaccines at the time indicated by health agencies. However, the results showed that when parents felt pity for their children receiving shots, they delayed the vaccination for at least 20 days. Since families visit the health center at least seven times before children complete five years of age and since most of the children does not take the measles vaccine in the exactly recommended day, but delay or anticipate the shots, it is clear that there is no compliance to the recommended government\'s measles vaccination schedule (first dose at 9 and second at 15 months of age), although this is the message that annual communication campaigns and interpersonal communication try to pass

Fatores associados com falhas na cobertura da vacinação contra o sarampo na cidade de São Paulo foram estudados por meio de entrevistas com pais ou responsáveis por 122 crianças de até cinco anos. Observou-se que a população conhece a doença e identifica a vacina com a idéia de proteção, mas não cumpre o calendário de vacinação e atrasa as doses da vacina contra o sarampo. Idade, região de origem ou de residência, status familiar ou nível educacional não mostraram associação com o cumprimento do calendário obrigatório (uma dose da vacina contra o sarampo deve ser aplicada dos 9 aos 11 meses e a segunda, aos 15 meses). A televisão é o meio de comunicação que mais informa os entrevistados sobre as Campanhas Nacionais de Multivacinação e o cartão de vacinação foi identificado como uma das vias de comunicação utilizadas para informar sobre a vacinação fora das Campanhas, na rotina. No entanto, nenhum meio de comunicação identificado no estudo pôde, significativamente, promover a mudança de comportamento da indife rença para o compromisso com o cumprimento do calendário, ou seja, adesão. A única variável significativamente relacionada com o atraso na vacinação, de até 20 dias, foi o sentimento de dó de aplicar injeções nas crianças (p = 0,08). Crianças brasileiras têm de visitar o posto de saúde para receber, até cinco anos de idade, 19 vacinas, 12 até o primeiro ano. O estudo deixa claro que não há adesão ao calendário proposto para vacinação contra o sarampo, apesar de ser esta a proposição das Campanhas Nacionais de Multivacinação e da vacinação de rotina / Risk factors associated with failure to receive measles vaccine were studied in eight districts of São Paulo city, Brazil. Parents or carers of 122 children were interviewed about their perception and understanding about the disease anda about measles vaccination, a kind of knowledge that can be acquired through vaccination communication campaigns or from personal communication (from the doctors, nurses of the health care center or friends). Population description factors were also identified. Results analysis shows that neither age, region of origin or residence, marital status nor educational level were related to taking or not taking measles vaccines adequately. Most of the people remembered about having being informed about the last annual vaccination campaign by television, but no communication channel was significantly associated with vaccination status. Answers to questions about knowing the disease or not or knowing the vaccine or not, when analysed alone, did not show any association with taking measles vaccines at the time indicated by health agencies. However, the results showed that when parents felt pity for their children receiving shots, they delayed the vaccination for at least 20 days. Since families visit the health center at least seven times before children complete five years of age and since most of the children does not take the measles vaccine in the exactly recommended day, but delay or anticipate the shots, it is clear that there is no compliance to the recommended government\'s measles vaccination schedule (first dose at 9 and second at 15 months of age), although this is the message that annual communication campaigns and interpersonal communication try to pass