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In dieser Arbeit wurden Vektorimpfstoffe auf der Basis rekombinanter Vacciniaviren hinsichtlich ihrer Eignung zur Immunisierung gegen Infektionen mit Respiratorischen Synzytialviren (RSV) untersucht. Hierfür standen genetisches Material und Viruspräparationen des Respiratorischen Synzytialvirus des Rindes (BRSV, Stamm Odijk) sowie des Respiratorischen Synzytialvirus des Menschen (HRSV, Subtyp A2) sowie rekombinante Vacciniaviren MVA-HRSV-F bzw. MVA-HRSV-G zur Verfügung. Rekombinante MVA-Viren, welche die Gene der BRSV-Oberflächenproteine G und F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu), sowie Viren in welchen die Fremdgensequenzen durch Deletion wieder entfernt sind (Revertante Viren MVA-∆BRSV-F und MVA-∆BRSV-G), wurden gentechnologisch hergestellt. Alle rekombinanten MVA-Viren wurden molekular-virologisch charakterisiert und dienten zur Gewinnung und Prüfung von Testimpfstoffen im Tiermodell. Die Untersuchungen zeigen: 1. Alle neu konstruierten rekombinanten MVA-BRSV-Viren produzierten nach Infektion von Zellkulturen die erwünschten Zielantigene, die BRSV-Glykoproteine F und G. Für das durch MVA-Expression hergestellte BRSV-F-Glykoprotein konnte außerdem die biologische Funktionalität in einem Fusionstest in infizierten HeLa-Zellen nachgewiesen werden. 2. Die Charakterisierung der Genome aller MVA-BRSV- sowie MVA-HRSV-Vektorviren bestätigte die exakte Insertion der Fremdgensequenzen im anvisierten Genombereich und zeigte die genetische Stabilität der Virusisolate nach Passagierung. 3. Bei der Untersuchung des Wachstumsverhaltens von MVA-BRSV-F und MVA-BRSV-G zeigte sich die eingeschränkte Vermehrungsfähigkeit des Virus MVA-BRSV-G. Die Konstruktion und Untersuchung der revertanten Viren MVA-∆BRSV-F und MVA-∆BRSV-G belegte die Koproduktion des G-Proteins als Ursache des verminderten Replikationsvermögens. Dieser für ein mögliches Impfvirus erhebliche Nachteil konnte durch die Verwendung eines moderateren Vacciniavirus-Promotors zur Fremdgenexpression (rekombinantes Virus MVA-BRSV-Gneu) behoben werden. 4. Die Prüfung von Testimpfstoffen auf der Grundlage der rekombinanten MVA-HRSV-Viren in einem Maus-HRSV-Infektionsmodell zeigte, dass MVA-HRSV-Impfstoffe, im Gegensatz zu Impfstoffen aus mit Formalin-inaktiviertem HRSV, Immunantworten mit einem ausgewogenen TH1/TH2-assoziierten Zytokinprofil induzierten. Eine infolge von Immunisierung verstärkte Einwanderung eosinophiler Zellen (Marker für Immunpathogenese) in die Lungen HRSV-infizierter Tiere, konnte nach MVA-Impfung nicht beziehungsweise in nur sehr geringem Ausmaß festgestellt werden (OLSZEWSKA et al. 2004). 5. Wichtige erste Daten hinsichtlich der Verträglichkeit, Immunogenität und Schutzwirkung rekombinanter Impfstoffe auf der Basis von MVA-BRSV-F und MVA-BRSV-G konnten in einem Kälber BRSV-Infektionsmodell erhoben werden. Die zweimalige Immunisierung mit MVA-Impfstoff verlief bei allen Tieren ohne feststellbare Nebenwirkungen und die Anregung Vaccinia- bzw. BRSV-F-spezifischer Antikörper bestätigte die Immunogenität der Vektorvakzinen. Schließlich belegten klinische Daten, insbesondere die fehlende Fieberreaktion bei Impflingen nach BRSV-Belastungsinfektion, die Schutzwirkung der MVA-BRSV-Impfstoffe. Insgesamt unterstützen die erzielten Ergebnisse dieser Arbeiten die weitere präklinische und klinische Untersuchung von MVA-Vektorimpfstoffen zur wirksameren und sichereren Bekämpfung von Infektionen mit Respiratorischen Synzytialviren. / This study investigated vector vaccines based on recombinant vaccinia virus MVA for their suitability to immunize against infections with respiratory syncytial viruses. Genetic material and virus stocks of bovine respiratory syncytial virus (BRSV, Strain Odijk) and human respiratory syncytial virus (HRSV, Strain A2) and recombinant vaccinia viruses MVA-HRSV-F and MVA-HRSV-G were provided and used in this study. The project work included the genetical engineering of recombinant MVA expressing gene sequences encoding the BRSV surface proteins G and F (MVA-BRSV-F, MVA-BRSV-G, MVA-BRSV-Gneu) and the secondary generation of mutant viruses in which recombinant gene sequences have been removed (revertant viruses MVA-∆BRSV-F, MVA-∆BRSV-G). All recombinant MVA were carefully characterized in in vitro experiments and served for generation of vaccine preparations being tested in animal model systems. The investigations demonstrate: 1. All recombinant MVA-BRSV viruses produced the target antigens (BRSV-F and -G proteins) upon tissue culture infections. Functional activity of BRSV-F protein was demonstrated in a cell fusion assay using virus-infected HeLa cells. 2. The characterization of the genomes of all MVA recombinant viruses confirmed the correct insertion of foreign gene sequences into the target site of the MVA genome and demonstrated the genetic stability of the vector viruses upon tissue culture passage. 3. In vitro studies on virus growth revealed a reduced replicative capacity of the recombinant virus MVA-BRSV-G. Construction and growth analysis of revertant viruses MVA-∆BRSV-F and MVA-∆BRSV-G demonstrated that over expression of BRSV-G protein caused this replication deficiency which could be avoided by using a more moderate vaccinia virus promoter for transcriptional control of recombinant gene expression (recombinant virus MVA-BRSV-Gneu). 4. Upon characterization in a mouse-HRSV challenge model candidate vaccines based on recombinant MVA-HRSV viruses, in contrast to formalin inactivated HRSV, and induced a well balanced TH1 and TH2 cytokine profile. In addition, none of the MVA-HRSV-F vaccinated animals and only two of the MVA-HRSV-G immunized mice showed low-level eosinophilia in the lungs after HRSV challenge infection (OLSZEWSKA et al. 2004). 5. Vaccination experiments in the calf-BRSV challenge model generated first relevant data on safety, immunogenicity and protective capacity of MVA-BRSV recombinant vaccines. The repeated application of MVA vaccine was well tolerated by all vaccinated animals and the induction of vaccinia- and BRSV-F-specific antibody responses confirmed the immunogenicity of the MVA vector vaccines. Moreover, clinical data (lack of fever response in vaccines) suggested the protective capacity of MVA-BRSV immunization upon BRSV challenge. The obtained results from these studies clearly support further preclinical and clinical evaluation of recombinant MVA candidate vaccines to immunize against disease caused by RSV infections in cattle and humans.

Magistro darbo objektas – interaktyvūs ryšių su visuomene įrankiai. Darbo tikslas – įvardyti interaktyvių ryšių su visuomene įrankių efektyvumo vertinimo metodus bei ištirti jų pritaikomumą praktikoje. Pagrindiniai uždaviniai, siekiant užsibrėžto tikslo, yra šie: 1) Apibrėžti interaktyvių ryšių su visuomene įrankių sąvoką, savybes bei tipus; 2) Išanalizuoti šių įrankių efektyvumo vertinimo metodus; 3) Tyrimo metu nustatyti šių įrankių efektyvumo vertinimo praktines galimybes. Siekiant tiksliai identifikuoti darbo objektą, pirmoje dalyje apibrėžiama interaktyvių ryšių su visuomene įrankių samprata bei tipai. Čia taip pat analizuojama, kaip sėkmingai juos panaudoti RSV veikloje. Antrame šio darbo skyriuje analizuojami efektyvumo vertinimo metodai, juos skirstant į kiekybinius bei kokybinius. Šioje dalyje nuosekliau išanalizuojama internetinio turinio stebėjimo metodika, kuri ir pritaikoma atliktame tyrime. Tyrimo rezultatai pristatomi trečioje darbo dalyje. Interaktyvūs ryšiai su visuomene – tai aktyvi organizacijos veikla, siekiant dialogo su publika. Tačiau tam tinkamiausi įrankiai – internetiniai dienoraščiai bei forumai. Kiti įrankiai, tokie kaip naujienų grupės, priimtini stebint pranešimus, kuriais keičiasi publika. Efektyvumo vertinimas taip pat priklauso nuo konkretaus naudojamo įrankio. Kiekybiniai vertinimo metodai – lankomumo analizė, grįžtamųjų nuorodų skaičius – leidžia išmatuoti matomumą, kurį įtakoja pasirinktas įrankis. Kokybiniai metodai leidžia vertinti... [toliau žr. visą tekstą] / The development of information and communication technologies has implications of many kinds in the everyday work of public relations (PR) practitioners. Web sites, e-mail, intranets, Internet forums and blogs have changed the way organization communicates with it‘s publics. These technologies give a lot of opputrtunities for PR specialist while implementing interactive communication with target audiences. The use of new communication technologies has added to communication within an organisation and between organisations the dimension of dialogue of many-tomany (e.g. a large number of dialogues in a discussion group, chat room or blog. It is difficult to control all the sources in wich the message about the oeganization can appear. „Times“ named the person of the year 2006 everyone, who uses Internet. That‘s how the traditional media shows, how customers generated content of the Internet has changed it‘s role of „gateskeaper“. Message in a forum or blog often becomes a message in a newspaper or a TV. But survey carried by European Public Relations Education and Research Association showed, that such tool as blog is heavily used by PR professionals PR specialists don’t see the main advantage of this technology. Another problem in adapting interactive PR tools for everyday work is ability to measure its effectiveness. It is difficult to evaluate the use of PR tool. The subject of research is interactive public relations tools. The aim of this research is to analyze the main... [to full text]

Šiandien komunikacijai su tikslinėmis auditorijomis įmonės vis dažniau naudoja interneto svetainę – masiškiausią reprezentacijos ir visuomenės informavimo būdą, taupantį komunikavimo laiką ir kaštus, gerinantį klientų aptarnavimą, leidžiantį skverbtis į naujas rinkas ir siūlyti naujas paslaugas. Rinkodaros naudojimas organizacijos interneto svetainėje padeda jai skatinti pardavimus, reklamuotis, stiprinti organizacijos ir prekinių ženklų žinomumą, konkuruoti su kitomis įmonėmis, didinti vartotojų lojalumą. Ryšiai su visuomene (toliau – RSV) svetainėje palaiko santykius su tikslinėmis auditorijomis, informuoja apie prekes ir įmonės veiklą, didina paklausą ir matomumą, mažina rėmimo kaštus, kuria teigiamą organizacijos įvaizdį. Tačiau daugelis įmonių savo svetainėse tinkamai neišnaudoja RSV ir rinkodaros galimybių. Darbo objektas – Lietuvos prekybos įmonių asociacijos (toliau – LPĮA) narių interneto svetainės. Darbo tikslas – analizuoti internetinės rinkodaros tipus, ištirti ir aprašyti interneto svetainių kaip RSV ir rinkodaros priemonių naudojimą prekybos įmonių komunikacijoje. Siekiant užsibrėžto tikslo, iškelti šie darbo uždaviniai: analizuoti naujųjų komunikacijos technologijų teikiamas galimybes ir interneto svetainių panaudojimą įmonės komunikacijos efektyvumui didinti; nustatyti LPĮA narių interneto svetainių tipus tiriant jose naudojamus rinkodaros ir RSV elementus; apklausus įmones, išskirti jų komunikavimo per svetainę ypatumus. Darbo metu atliktas kiekybinis tyrimas... [toliau žr. visą tekstą] / Corporate Web site – is the most massive way of representation and public information, saving time and communication costs, enhancing customer service, which allows the penetration of new markets and offer new services. The use of marketing in corporate web site helps to promote sales, strengthen the brand awareness, advertise, compete with other companies, strengthen consumer loyalty. Public Relations (hereinafter – PR) in corporate web site maintains relationships with target audiences, informs about products and business activities, increases demand and visibility, reduce support costs, creates positive image of the organization. Work item – website of the Lithuanian Trade Enterprises Association (hereinafter – LTEA) members. The aim of the work – to analyze the types of internet marketing, explore and describe the web as the PR and marketing tool in communication of trading company. In order to seek the target, the following work tasks were raised: to analyse the impact of the new communication technologies and the use of coporate Web site to increase the efficiency of communication; to determine the types of LTEA members Web sites exploring the use of marketing and RSV elements, interviewing the organizations, to identify their communication through the site features. Raised hypotheses of the work: the web site – the most effective PR and marketing tool of the new century; Web sites of the LTEA members are more orientated into produkcts. Working on a quantitative survey... [to full text]

Magistro darbo objektas – ryšių su visuomene pranešimai spaudai. Darbo tikslas – išsiaiškinti, koks žiniasklaidos vaidmuo ryšių su visuomene veikloje, ir nustatyti, kaip atsiskleidžia naujienos ir reklamos santykis RsV pranešimuose. Pagrindiniai darbo uždaviniai: aptarti ryšių su visuomene veiklos principus; nustatyti RsV pranešimų ypatybes ir rašymo pagrindus; nurodyti, kokie yra svarbiausi naujienos kriterijai; pastebėti, kaip pranešimuose naudojami reklamos elementai; išsiaiškinti, kaip informacija turi būti pateikiama žiniasklaidai; suprasti, kaip ji publikuojama visuomenės informavimo priemonėse; pastebėti, kas įtakoja informacijos pateikimą visuomenei. Naudojantis dokumentų ir regioninių laikraščių redaktorių apklausos analize, prieita prie išvados, kad ryšių su visuomene pranešimuose ne tik informuojama, bet yra skelbiama ir organizacijos reklama. Kadangi pastebimas žiniasklaidos poveikis informaciją gaunančiai visuomenei, pranešimai spaudai yra populiariausia ir dažniausiai naudojama ryšių su visuomene bendravimo forma. Tačiau tikslas informuoti visgi dažniau siekiamas su noru įtikinti ar paveikti. Tuo tarpu RsV specialistai privalo suprasti, kad reikia platinti pranešimus žiniasklaidai ir juose pateikti tik esmę ir naudoti svarbiausius naujienų kriterijus, reikalingus žurnalistiniam tekstu. Išryškėja tendencijos, jog ir žiniasklaidos priemonėse visuomenei žinios pateikiamos ne objektyviai ir dažnai veikia įtikinimo elementai. Regioninių laikraščių redaktorių... [toliau žr. visą tekstą] / Final work „Press release in public relations“ analyses the press release position and its importance in modern society. The main purpose of this work – to find out the role of mass media in public relations and determine real relationship between news and advertisement in press releases. Main tasks, determined in the final work are: to dispute activity fundamental of public relations; to define peculiarities and writing instructions of press release; to indicate the main news criteria; to observe advert details in press releases; to find out how information should be offered to mass media; to understand how it is published in mass communication; to find out influence groups, that prescribe published information to society. Conclusions can be drawn: press releases not only inform, but also declare organization’s advertisement in public relations. Mass media impact to society is observable, that’s why press releases are the most popular and commonly usable in public relations. However the intention to inform is switching with desire to persuade or impress. In the mean time specialists of public relations must understand it’s need to distribute press releases and give only the main point – use prime news criteria like journalists. Tendencies are showing up that society is getting unfair information with persuasion details from mass media. Analysis of local mass media editors’ poll have indicated, that promotion men and public relations specialists influence publications while... [to full text]

Les infections aiguës des voies respiratoires inférieures constituent la troisième cause de décès dans la population mondiale, avec 3,2 millions de décès. Parmi cette mortalité, au tour de 1 million c’était des enfants de moins de 5 ans, représentant la première cause de mortalité dans ce groupe d’âge, selon l'OMS en 2015. Le virus respiratoire syncytial (VRS) est considéré comme un agent étiologique important dans le millieux pediatric et les estimations sont que ce virus cause 3 million d’hospitalization par an. Un aspect important du pronostic des infections virales est le rôle de co-infection bactérienne. La combinaison d’agents viraux et bactériens a été signalée entre le VRS et les bactéries Streptococcus pneumoniae. En raison de l’importance clinique de cette co-infection et le taux élevé de circulation du VRS, il est important de comprendre comment le système immunitaire est affecté à l'infection de ces deux agents pathogènes. Notre étude identifie la réponse immunitaire dans les macrophages, ainsi comme les interactions entre le VRS et le facteur de transcription p53. Les résultats montrent un profil particulier a cette co-infection mixte dans le macrophages et des modifications dans la réponse immune innée que nous a permis de mieux comprendre les mécanismes de pathogenèse du VRS dans les cellules épithélial pulmonaires en regardant la régulation de p53. Dans la dernière partie, nous avons évalué l'impact direct de l’infection mixte chez les primates non-humain et ce modèle nous a montré les difficultés et complexités des établir une pneumonie sévère. / Respiratory viruses play a leading role in the etiology of respiratory infections. Currently, respiratory syncytial virus (RSV) is generally considered to be the etiologic agent of respiratory disease in pediatric importance, as children can develop bronchiolitis and pneumonia when infected with the virus. The first RSV infection occurs in the first two years of life in about 95% of children, with the peak incidence occurring in the first few months of life. An important aspect of the prognosis of viral infections is the role of bacterial co-infection. The combination of viral and bacterial agents has been reported between RSV and Streptococcus pneumoniae bacteria. Because of the clinical importance of this co-infection and the high rate of RSV circulation, it is important to understand how the immune system is affected by the infection of both pathogens. Our study was designed to evaluate the immune response in macrophages, in addition to interactions between RSV and p53 transcription factor. The results show a particular profile of this mixed co-infection in macrophages and p53 regulation that implies several modifications in the innate immune response and that allowed us to better understand the mechanisms of pathogenesis of RSV in pulmonary epithelial cells. In the last part, we evaluated the direct impact of mixed co-infection in non-human primates and this model showed us the difficulties and complexities of establishing severe pneumonia.

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling has a pivotal role in the spread of RSV infection.

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<p> Proper gene expression programs cellular activities, while aberrant manipulation of transcription factors often leads to devastating consequences, such as cancer or cell death. The transcription factor family activator protein-1 (AP-1) plays an important role in many cellular activities including cell transformation, proliferation and survival (Shaulian and Karin 2002). However, little has been done to obtain a global view of the role of individual AP-1 members and how they cooperate in many cellular activities. We have discovered that blocking the AP-1 pathway by a c-Jun dominant negative mutant, TAM67, induced cell death in RSV-transformed primary chicken embryo fibroblasts (CEF), suggesting that AP-1 activity is vital for cell survival upon v-Src transformation. In addition, accumulation of cytoplasmic vesicles was observed in the cytoplasm of a proportion of RSV-transformed CEF expressing TAM67. Oil-red staining of these vesicles indicated the presence of lipid droplets in these cells, suggesting that the inhibition of AP-1 promotes the adipogenic conversion of v-Src transformed CEF. To understand the role of individual members of the AP-1 family, a retroviral-based shRNA expressing system was designed to stably downregulate individual AP-1 members. This retroviral-based RNAi system provided sustained gene downregulation of AP-1 family members. Reduction of the c-Jun protein level by shRNA induced senescence in normal CEF, while it modestly downregulated AP-1 activity in RSV -transformed CEF indicating that c-Jun is not the main component of the AP-1 complex in RSV-transformed CEF. Inhibition of JunD expression induced apoptosis and was deleterious to both normal and RSV-transformed CEF, suggesting that JunD is crucial for the survival of CEF. Transient express10n reporter-assays also showed that loss-of-function of JunD by shRNA dramatically repressed AP-1 activity. Hence JunD is the main component of the AP-1 complex that regulates the survival of CEF. Furthermore, we determined that loss of JunD expression resulted in an elevated level of tumour suppressor p53. Co-inhibition of p53 and JunD restored the transforming ability of v-Src transformed CEF, as indicated by foci formation in soft agar assays. Hence, repression of p53 induction was able to bypass the death signal released as a result of AP-1 inhibition in v-Src transformed CEF. Downregulation of Fra-2 (Fos-related antigen 2) level by shRNA did not affect the proliferation of normal CEF. However, RSV -transformed CEFs expressing fra -2 shRNA were transformation-defective with the presence of multiple vesicles in cytoplasm. Oil-red staining of these vesicles indicated the presence of lipid droplets, which resembles the effect of T AM67 in RSV -transformed CEF indicating that Fra-2 blocks differentiation. These findings help us to understand the role of individual members of the AP-1 transcription factor family in normal and RSV -transformed CEF. Importantly, global gene profiling of v-Src transformed CEF expressing shRNA for individual AP-1 members will improve our knowledge of the transformation process. Functional characterization of the cascade will rely on the use of retroviral-based shRNA expressing system as described above. </p> / Thesis / Doctor of Philosophy (PhD)