Submitted by (lucia.rodrigues@ufrpe.br) on 2016-07-05T12:16:24Z No. of bitstreams: 1 Emmanuella Vila Nova da Silva.pdf: 1939037 bytes, checksum: f521d3bf721007766ec42ccf2f7454a6 (MD5) / Made available in DSpace on 2016-07-05T12:16:24Z (GMT). No. of bitstreams: 1 Emmanuella Vila Nova da Silva.pdf: 1939037 bytes, checksum: f521d3bf721007766ec42ccf2f7454a6 (MD5) Previous issue date: 2012-03-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The use of plants symbiotically associated with N2 fixing bacteria and mycorrhizal fungi (AMF) provides an efficient strategy to accelerate the recovery of impacted areas and reduces its costs considerably. The term "mycorrhiza helper bacteria” (MHB) has been introduced and discussed due to the synergistic effect that this dual combination promotes to plants. They are bacteria associated with roots and AMF that selectively promote the establishment of symbiosis with fungi. Thus, the objectives were to verify the AMF activity in the area with native vegetation in the Pernambucano semiarid, municipality of Sertânia; determine glomerospores number and the most probable number (MPN) of infective propagules; quantify the content of glomalin-related protein in the soil and determine the feasibility of bacteria (MHB) co-inoculation and AMF mixture in “sabiá” (Mimosa caesalpiniifolia Benth) aiming at obtaining combinations and compatibility of symbiotic pairs, as well as to evaluate the mycorrhizal efficiency and colonization. The experiments were conducted in greenhouse of the Agronomic Institute of Pernambuco (IPA). 10 composite soil samples were collected with points were defined at random. Samples were homogenized and analyzed for physical and chemical characteristics. Composite samples were used for direct count (DC) and propagation of AMF for indirect count (IC) of spores, using trap- cultures and sorghum (Sorghum bicolor L. Moench) and peanut (Arachis hypogea L.) as host plants (experiment I). To determine the MPN of infective propagules of AMF in the Haplic Luvisol was used a system of serial dilution: 0, 1:10, 1:100 and 1:1000 with five replicates each, with maize (Zea mays L.) as host plant (experiment II). In the experiment III were used pots with Haplic Luvisol soil (8 kg pot-1) at pH 6.0 and the plant used was the “sabiá”. On seeding, inoculation with Burkholderia sabiae (BR 3405) and co-inoculation with BR3405 + MHB were performed and each seed was inoculated with 2 mL of specific medium for each of MHB bacteria and for the BR3405 containing 108 CFU mL-1. In the inoculation with AMF mixture was used 4 g pot-1 in the form of propagule containing approximately 670 spores. Plants were harvested at 110 days after planting (DAP) and the following variables were evaluated: shoot dry mass (SDM), root (RDM), RDM/SDM ratio, plant height (PH) on periods of 45, 90 and 110 days, root length (RL), total N accumulated in SDM (Nat), strains efficiency (E) and mycorrhizal colonization. The experimental design was randomized blocks, with 9 x 2 factorial arrangement plus an absolute control (AC) - without inoculation; MHB strains and one control treatment inoculated only with Burkholderia sabiae with and without AMF (AMF mixture) with 3 blocks . The experimental results show that the MPN of AMF infective propagules found in the city of Sertânia was 23 propagules cm-3. Soil proteins related to easily extractable glomalin (PSRGFE) and soil proteins related to total glomalin (PSRGT) were approximately 0.46 and 0.26 mg g soil-1, respectively. The AMF colonization combined with the bacteria was positive, as in the case of RL, treatments with BR 3405 + Azospirillum amazonenses (Y2) and BR 3405 + Herbaspirillum seropedicae (Z67) showed significant difference by the Tukey test (p <0.05) compared to the factor with and without AMF. Thereby ensuring that, in the presence of MHB bacteria there was increase in root length of “sabiá” plants. Strains efficiency showed better results when bacteria were in the presence of AMF and the treatment BR 3405 + Paenibacillus brasilensis (24) + AMF showed the best response. The treatments that received AMF were higher compared to the others on the variables SDM, RDM, E, Nac, coming to present on average 84% of root colonization. / A utilização de plantas associadas simbioticamente, com bactérias fixadoras de N2 e fungos micorrízicos arbusculares (FMA), constitui uma estratégia eficiente para acelerar a recuperação de áreas impactadas além de reduzir consideravelmente os custos com a mesma. O conceito de “mycorrhiza helper bacteria (MHB)” tem sido introduzido e discutido devido ao efeito sinergístico que essa dupla associação promove às plantas. São bactérias associadas com raízes e FMA que, seletivamente, promovem o estabelecimento da simbiose com os fungos. Deste modo, os objetivos deste trabalho foram verificar a atividade de FMA em área com vegetação nativa do semiárido Pernambucano, no município de Sertânia; determinar o número de glomerosporos e o número mais provável (NMP) de propágulos infectivos; quantificar o teor de proteínas do solo relacionadas à glomalina; determinar a viabilidade da co-inoculação entre bactérias (MHB) e mistura de FMA em sabiá (Mimosa caesalpiniifolia Benth) visando obter combinações e compatibilidade de pares simbióticos, assim como avaliar a eficiência e colonização micorrízica. Os experimentos foram conduzidos em casa de vegetação na Sede do Instituto Agronômico de Pernambuco (IPA). Foram coletadas 10 amostras compostas de solo, sendo os pontos definidos aleatoriamente. As amostras foram homogeneizadas e analisadas quanto às características físicas e químicas. Amostras compostas foram utilizadas para contagem direta (CD) e multiplicação de FMA para contagem indireta (CI) de esporos, com o uso de culturas-armadilha, empregando sorgo granífero (Sorghum bicolor L. Moench) e amendoim (Arachis hypogea L.) como plantas hospedeiras (experimento I). Para a determinação do NMP de propágulos infectivos de FMA no Luvissolo Háplico foi utilizado um sistema de diluição em série: 0, 1:10, 1:100 e 1:1000, com 5 repetições cada e, tendo o milho (Zea mays L.) como planta hospedeira (experimento II). No experimento III foram utilizados vasos com o solo Luvissolo Háplico (8 kg vaso-1) com pH 6,0 e a planta utilizada foi a sabiá. Na semeadura, foi efetuada inoculação com Burkholderia sabiae (BR 3405) e co-inoculação com BR3405 + MHB contendo 108 UFC mL-1. Na inoculação com a mistura do FMA, foram utilizados 4 g vaso-1 em forma de propágulo, contendo aproximadamente 670 esporos. A colheita foi realizada 110 dias após plantio (DAP) e foram avaliadas as seguintes variáveis: massa seca da parte aérea (MSPA), raiz (MSR), relação MSR/MSPA, altura de planta (AP) nos períodos de 45, 90 e 110 dias, comprimento da raiz (CR), N total acumulado na MSPA (Nac), eficiência das estirpes (E%) e colonização micorrízica. O delineamento experimental adotado foi em blocos casualizados, com arranjo fatorial 9 x 2 mais uma testemunha absoluta (TA) – sem inoculação; estirpes de MHB e um tratamento controle inoculado apenas com Burkholderia sabiae com e sem FMA (mistura de FMA) com 3 blocos. Os resultados dos experimentos mostram que o NMP de propágulos infectivos de FMA encontrados no município de Sertânia foi de 23 propágulos cm-3. As proteínas do solo relacionadas à glomalina facilmente extraível (PSRGFE) e as proteínas do solo relacionadas à glomalina total (PSRGT) ficaram em torno de 0,46 e 0,26 mg g solo-1, respectivamente. A colonização dos FMA em conjunto com as bactérias foi positiva, como no caso do CR, os tratamentos com BR 3405 + Azospirillum amazonenses (Y2) e BR 3405 + Herbaspirillum seropedicae (Z67) apresentaram diferença significativa pelo teste de Tukey (p<0,05) em relação ao fator com e sem FMA. Confirmando deste modo que, na presença das bactérias MHB houve aumento no comprimento do sistema radicular das plantas de sabiá. A eficiência das estirpes obteve os melhores resultados quando as bactérias estavam em presença de FMA e o tratamento BR 3405 + Paenibacillus brasilensis (24) + FMA foi o que obteve melhor resposta. Os tratamentos que receberam os FMA foram superiores em relação aos demais nas variáveis MSPA, MSR, E, Nac, chegando a apresentar uma média em torno de 84% de colonização radicular.

La réponse humorale est altérée lors de l’infection par le virus de l’immunodéficience humaine (VIH). Les lymphocytes T CD4+ folliculaires helper (Tfh) sont impliqués dans la maturation des lymphocytes B (LB) dans les organes lymphoïdes secondaires. Mon travail de thèse s’est articulé autour de deux axes complémentaires visant à étudier les Tfh et les réponses B mémoires lors de l’infection par le VIH. J’ai d’abord étudié les Tfh spléniques lors de la phase chronique de l’infection par le VIH. J’ai mis en évidence une augmentation des populations de Tfh dans les rates de patients VIH+. D’autre part l’infection par le VIH a un impact sur le profil transcriptionnel des Tfh de la rate et la production de cytokines impliquées dans la différenciation des LB, suggérant un défaut fonctionnel des Tfh. Parallèlement, la maturation des LB est altérée dans les rates VIH+. Dans le second axe de ma thèse, j’ai étudié les réponses B mémoires anti-VIH dans différentes cohortes de patients VIH+ : Elite controller (EC) contrôlant l’infection sans traitements et des patients VIH+ traités. J’ai mis en évidence que les EC préservent naturellement leurs compartiments B mémoires et que les réponses B mémoires spécifiques du VIH sont maintenues dans le sang de ces patients. Les réponses B mémoires IgG1+ anti-VIH sont majoritaires chez les EC, tandis que les réponses IgG2+ et IgG3+ sont plus rares. Ces travaux permettent une meilleure compréhension de la physiopathologie de l’infection par le VIH en apportant de nouveaux éléments sur la fonctionnalité des Tfh et les réponses B mémoires anti-VIH. / HIV infection is associated with a defect of humoral response. T follicular helper cells (Tfh) support multiple steps of B cell maturation and antibody production. My work was divided in two complementary axes aiming to characterize Tfh and memory B cell responses in HIV-infected patients.I identified several Tfh populations in HIV+ and HIV- spleens by FACS. These three populations were increased in HIV+ spleen. I also evidenced an impact of HIV infection on transcriptional profile and a compromised production of B cell differentiation-related cytokines by splenocytes from HIV+ donors. These results suggest Tfh functions impairment during HIV-infection. In parallel, we noticed an altered maturation of B cells in HIV+ spleens. In a cohort study, we compared memory B cell responses in the blood of Elite controllers (EC) who naturally control HIV and treated HIV+ patients. I evidenced that EC naturally preserve their memory B cell compartments. In contrast to anti-HIV IgG2 and IgG3 secreting B cells, most EC exhibit a high frequency of anti-HIV IgG1 secreting B cells. My work highlights a defective Tfh differentiation, which might explain why B cell maturation is severely affected in HIV-progressors. The status of HIV-controller seems associated with the presence of an IgG1 B cell memory response. Further work will highlight whether Tfh functions are preserved in EC.

La nature des cellules dendritiques (DC) engagées lors d'une vaccination conditionne la qualité de la réponse immunitaire adaptative. L'immunisation par la peau est particulièrement efficace car elle cible de nombreuses sous-populations de DC cutanées telles que les cellules de Langerhans (LC). Cependant, les relations entre ces DC et les cellules effectrices associées à la réponse humorale ne sont pas connues. L'objectif de ma thèse est d'identifier les mécanismes cellulaires précoces impliqués dans l'initiation de la réponse humorale, dans un contexte de vaccination intradermique (i.d.) avec un antigène particulaire. En étudiant la distribution spatiale et temporelle des particules synthétiques de PLA adsorbées par la protéine p24 du VIH, nous avons observé leur prise en charge par les DC cutanées mais également par les DC résidentes des ganglions drainant de la peau. Cependant, l'étude de la réponse immunitaire a démontré que seules les cellules cutanées, et en particulier les LC, induisent la polarisation des lymphocytes T CD4+ folliculaires (TFH) et le développement des lymphocytes B sécrétant des IgA. L'immunisation i.d. a également généré l'infiltration de cellules inflammatoires au niveau du site d'injection et du ganglion. En utilisant un modèle murin Ccr2-/-, nous avons démontré que les cellules dépendantes de CCR2+ interfèrent avec la formation des TFH. Enfin, l'étude du micro-environnement ganglionnaire suggère que TNF est favorable à la polarisation des TFH. En conclusion, ces résultats soulignent l'importance de cibler les DC cutanées lors de la vaccination afin de proposer de nouvelles stratégies vaccinales. / The quality of the adaptive immune response to a vaccine is driven by the nature of dendritic cells (DCs) engaged during vaccination. Skin immunization is particularly efficient as it targets the numerous cutaneous DCs, including Langerhans cells (LCs). However, the relationship between DCs and effector cells associated with humoral immunity has not been elucidated. The main objective of my thesis was to identify cellular mechanisms implicated in the initialization of the humoral immune response, in the context of intradermal (i.d.) vaccination with particle-based antigens. In examining the spatial and temporal distribution of synthetic PLA particles adsorbed with the HIV-p24 protein, we observed their uptake by both cutaneous DCs and also skin-draining lymph node (dLNs) resident DCs. However, our immune response study highlighted that only skin cells, and in particular LCs, were able to stimulate polarization of follicular helper T cells (TFH) and the development of IgA-secreting B lymphocytes. I.d. vaccination also induced an inflammatory cell infiltration at both the injection site and in dLNs. Using a Ccr2-/- mouse model, we have shown the CCR2+ dependant cells can interfere in TFH polarization. Finally, the study of the dLN micro-environment suggested TNF can promote TFH formation. In conclusion, these findings highlight the importance of targeting skin DC in vaccination to propose new vaccine strategies.

In this thesis, Sebilwane is the subject of a narratological investigation. The point of departure of this study is based on the fact that a narratological text consists of three levels: the history, the composition, the usage of words which are recognisable in the style of the author. The epic-poem is not the subject of a verse-technical investigation and description. The narratological model is adapted to the aim of this study. The historical level regarded in principle as the original level prior to the material's exposure to a viewpoint and it is interpreted. The four narrative elements that are investigated are: the events, the characters/actors, time and place. In Sebilwane there are main events identified by the criteria of: (a) change, (b) cause, and (c) result. The characters/actors have been described and classified according to: (a) aim, (b) supporter, (c) patron, (d)helper and patron,and (e) opponent. In as far as historical time is concerned, it has been concluded that the events occurred: (a) in the remote past, and (b) stretched it to a 24 hour period. The actors/persons find themselves in a rural area which can be comparable to Botlokwa which is lying within the borders of Lebowa. The composition of the information which is given in the historical level, gives the shape of the author's aim. Here, what is important, are the functions which are described by the elements themselves. Then the idea of the theme comes clearly in this part and it is therefore identified as the main - and sub-theme. The third level concerns the usage of words; the information now gets a personal or subjective selection. Therefore, only a short passage is to be selected for stylistic analysis. The analytic model which is effected here is Kerkhoff's. AFRIKAANS : In hierdie verhandeling word Sebilwane aan 'n narratologiese ondersoek onderwerp. Die uitgangspunt van hierdie studie is dat 'n narratologiese teks uit drie lae bestaan: die geskiedenis, die samestelling, die verwoording wat in die styl van die outeur kenbaar is. Hierdie epiese gedig word nie verstegnies ondersoek en beskryf nie. Die narratologiese model is vir die doel van hierdie studie aangepas. Die geskiedenislaag word in beginsel as die oorspronklike laag beskou voordat die gegewens vanuit 'n bepaalde gesigspunt bekyk en weergegee word. Die vier vertelelemente wat ondersoek word, is die gebeurtenisse, die karakters/akteurs, tyd en plek. In Sebilwane is die kerngebeurtenisse geïdentifiseer deur die kriteria van (a) verandering, (b) oorsaak en (c) afloop. Die karakters/akteurs is beskryf en geklassifiseer volgens (a) doelstelling, (b) begunstigde, (c) begunstiger, (d) helper en (e) teëstaander. Wat die tyd betref, speel die gebeure (a) histories in die verre verlede af, en (b) strek dit oor 'n 24 uur tydperk. Die akteurs/mense bevind hulle in 'n landelike gebied wat waarskynlik Botlokwa is wat binne Lebowa geleë is. Die samestelling van die gegewens wat in die geskiedenislaag gegee is, gee aan die doestelling van die outeur gestalte. Daarvan gaan dit hier om die funksies wat aan die elemente toegesê word. Die begrip van die tema staan in hierdie gedeelte voorop, en daar word 'n hoof - en 'n subtema geïdentifiseer. / Dissertation (MA)--University of Pretoria, 1993. / African Languages / unrestricted

DNA immunization, inoculation with an antigen-expressing plasmid DNA, is a new method for generating an antigen-specific immune response. At the time these investigations began, very little was known about the immune response produced by DNA vaccines. Thus, the first aim of our studies was to perform a detailed examination of the antibody response generated by DNA immunization with an influenza hemagglutinin (HA)-expressing DNA in BALB/c mice. Using several different routes and methods of DNA immunization, we observed a number of findings. Although all three forms of DNA immunization elicited strong anti-HA antibody responses, i.m. and i.d. saline DNA immunization required approximately 100 times more DNA than a gene gun DNA immunization to raise an equivalent titer of anti-HA antibody. Indeed, as little as one inoculation and one boost by gene gun of 0.0004 μg of DNA produced a measurable antibody response in 50% of mice. Unexpectedly, we found the isotype of the antibody response differed among groups of mice immunized by different forms of DNA immunization. Intramuscular and i.d. saline DNA immunization produced predominantly an IgG2a anti-HA antibody response, whereas gene gun DNA immunization elicited mostly an IgG1 anti-HA antibody response. Considering that IgG2a and IgG1 antibody isotypes were known to correlate with Th1 and Th2 immune responses, respectively, we analyzed the type of immune responses produced by i.m., i.d., and gene gun DNA immunization. We found that i.m. and i.d. saline DNA immunization produced a Th1 predominant cellular immune response. In contrast, gene gun DNA immunization produced a Th2 cellular immune response. The differences in the type of immune responses were found to be due to the method of DNA immunization, and not due to the route of DNA inoculation. A gene gun DNA immunization of muscle produced the same IgG1, Th2 immune response as a gene gun DNA immunization of skin, while a saline DNA immunization of muscle and skin produced mostly an IgG2a, Th1 immune response. Each method of DNA immunization created good memory Th cell responses. The type of immune response created by an initial DNA immunization remained fixed even after multiple boosts with the identical method of DNA immunization, following a boost with the alternative method of DNA immunization, or after a viral challenge. The differentiation of naive Th cells into Th1 or Th2 cells depends on a variety of factors. We performed many experiments to elucidate which factors played a role in the generation of Th1 or Th2 immune responses following saline DNA immunization and gene gun DNA immunization. DNA dose response studies revealed the use of different doses of DNA between groups of saline DNA and gene gun DNA immunized mice did not account for the differentiation of distinct Th cell subsets. Cytokine production inducible by a number of factors inherently associated with either saline DNA or gene gun DNA immunization did not affect Th differentiation. For instance, contamination of plasmid DNA with lipopolysaccharide did not account for differences in the immune response. Immunostimulatory CpG sequences did not affect Th differentiation following DNA immunization, but they did enhance the IgG2a antibody response to coinoculated HA protein. Finally, cotransfection of IFNγ or IL-4 expressing plasmids with an HA-expressing plasmid by gene gun inoculation or as a saline DNA injection did not shift the type of immune response in a Th1 or Th2 direction, respectively. Thus, it appeared that increased cytokine stimulation was not responsible for selective Th subset differentiation. One factor related to the method of DNA immunization did seem to correlate with Th1 differentiation. Deposition of plasmid DNA extracellulary by saline DNA injections (as opposed to intracellular DNA delivery by gene gun) may have stimulated Th1 immune responses. Manipulating a gene gun DNA immunization to deliver DNA to the dermis (and thus extracellularly) shifted the immune response from that of a Th2 type to a mixed Th1/Th2 type. Furthermore, evidence was gathered demonstrating that pDNA can interact with cell surface molecules and that specific sequences in pDNA can act as a ligand and bind to molecules. Taken together, our data led us to propose a new model for Th1 differentiation following saline DNA immunization. We believe extracellular pDNA binds to an APC cell surface molecule which activates the cell. The activated APC preferentially stimulates naive Th cells to differentiate into Th1 cells. Finally, studies using a variety of mice differing in their genetic backgound and MHC genotype demonstrated the generality of our findings regarding i.m. saline DNA inoculations of an HA-expressing pDNA. Saline DNA immunization produced IgG2a, Th1-predominant immune responses independent of the genetic background and MHC genotype of the mice. In contrast, the type of immune response elicited by a gene gun DNA immunization was dependent on the MHC genotype of mice. Thus the type of immune response produced by gene gun DNA immunization probably depends on the specific antigen (and its effect on MHC-peptide/TcR interaction and signaling) and is less likely due to any inherent feature associated with the process of gene gun DNA delivery.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21451号 / 医博第4418号 / 新制||医||1032(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 椛島 健治, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

During the course of an immune response, CD4+ T helper cells differentiate into a number of subsets including: T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. The functional diversity of CD4+ T effector cells results in a coordinated, pathogen-specific immune response. For example, the production of IFNγ by TH1 cells is vital for the clearance of intracellular pathogens, while TFH cell engagement with cognate B cells is required for germinal center (GC) formation and the generation of pathogen- and vaccine- induced antibody production. The development of CD4+ subsets is contingent on extracellular signals, in the form of cytokines, and downstream transcriptional networks responsible for promoting the unique gene expression profile for each subset while simultaneously suppressing alternative cell fates. However, the exact composition of, and stage-specific requirements for, these environmental cytokines and transcription factor networks in the governance of TFH cell differentiation remain incompletely understood. The work in this dissertation seeks to understand how cell-extrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Here, we demonstrate that in response to decreased IL-2 and constant IL-12 signaling, T helper 1 (TH1) cells upregulate a TFH-like phenotype, including expression of the TFH lineage defining transcription factor Bcl-6. Intriguingly, our work established that signals from IL-12 were required for both the differentiation and function of this TFH-like population. Mechanistically, IL-12 signals are propagated through both STAT3 and STAT4, leading to the upregulation of the TFH associated genes Bcl6, Il21, and Icos, correlating with increased B cell helper activity. Conversely, exposure of these TFH-like cells to IL-7 results in the STAT5-dependent repression of Bcl-6 and subsequent inhibition of the TFH phenotype. Finally, we describe a novel regulatory mechanism wherein STAT3 and the Ikaros zinc finger transcription factors Ikaros and Aiolos cooperate to regulate Bcl-6 expression in these TFH-like cells. Collectively, the work in this dissertation significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines. / Ph. D. / Specialized cells called T helper cells serve as a critical interface between the innate (first line of defense) and adaptive (specialized and long-term) immune systems. During the course of an infection, T helper cells are responsible for orchestrating the immune-mediated elimination of invading viruses, bacteria, and parasites. This wide breadth of functionality is achieved through the formation of distinct T helper subsets including T helper 1 (TH1), TH2, TH17, and T follicular helper (TFH) populations. Individual subsets have distinct developmental requirements and have unique functions within the immune system. For example, TFH cells are required for the production of effective antibodies that recognize invading pathogens, leading to their subsequent elimination. This naturally occurring process is the basis for a number of modern medical therapies including vaccination. Conversely, aberrant generation of antibodies that recognize host tissues can result in the onset of various autoimmune diseases including lupus, multiple sclerosis, and crohn’s disease. Due to the importance of TFH cells to human health, there is intense interest in understanding how these cells are formed. It is recognized that the generation of these therapeutically important immune cells is mediated by numerous cell-extrinsic andintrinsic influences, including proteins in their cellular environment called cytokines, and important proteins inside of the cell called transcription factors. However, as this is a complicated and multi-step process, many questions remain regarding the identity of these cytokines and transcription factors. The work in this dissertation seeks to understand how cellextrinsic cytokine signals and cell-intrinsic transcription factor activities are integrated to properly regulate TFH cell development. Collectively, this body of work significantly advances our understanding of the regulatory mechanisms that govern TFH cell differentiation, setting the basis for the rational design of novel immunotherapeutic strategies and increasingly effective vaccines.

This diploma thesis is focused on the use of mobile devices in chemistry education. Describes various mobile devices, including different operating systems and technology and shows huge potential that these devices bring to education. It also includes an overview of existing educational programs with a chemical theme on mobile devices. Part of this work was to create a custom supportive program Chemical helper for mobile devices, which can be used in chemistry education, laboratory and also in everyday life.

O Adenovírus (Ad) é um agente etiológico que causa infecções em diversas espécies e também pode ser utilizado na forma de vetor como ferramenta tecnológica para terapia gênica. O Controle sobre a replicação de Ad pode trazer beneficio para o combate de infecções e para as tecnologias de transferência genica. Porém, poucas ferramentas existem que podem inibir a replicação de Ad. Uma aplicação importante seria a inibição da replicação de Adenovírus helper utilizado na produção de Vírus Adenoassociado recombinante (rAAV), assim minimizando contaminação da produção de rAAV com o virus helper. Dessa maneira o objetivo desse trabalho foi investigar se há inibição da replicação do Ad mediada por RNA de interferência (RNAi) direcionada para alvos adenovirais chaves. Para isso foram construídos vetores lentivirais que codificam shRNAs para os genes hexon, IVa2 e pol. Em seguida foram criadas linhagens que expressam constitutivamente os shRNAs em 293T, células onde os vetores adenovirais conseguem se replicar. Os shRNAs específicos para hexon e IVa2 promoverem significantemente a redução dos níveis destes mRNAs conforme revelado utilizando RT-qPCR para quantificação dos transcritos adenovirais. Em seguida, knockdown do gene hexon se mostrou promissor em inibir a replicação do Ad, visto como redução de vírus produzido em células 293T anti-hexon. O knockdown do transcrito de hexon e a redução em replicação de Adenovírus foram mais acentuados após cell sorting e obtenção de clones celulares a partir da linhagem anti-hexon. O clone anti-hexon mostrou significante redução na quantidade de partículas adenovirais visualizadas por microscopia eletrônica e redução de 92% das partículas infecciosas em relação a 293T quando a produção foi realizada em larga escala. Esses resultados indicam que a tecnologia de shRNA para inibir a replicação do Ad é promissora e representa o primeiro passo de desenvolvimento de uma estratégia para a produção de rAAV livre de contaminação com Ad helper / Adenovirus (ad) is an etiologic agent that causes infections in diverse species and can also be used as a technologic resource, such as a vector applied in gene therapy. Control over Ad replication could be beneficial for the combat of infections and for the technology of gene transfer. However, few tools exist that may useful for the inhibition of Ad replication. One important application would be to impede replication of helper adenovirus utilized in the production of recombinant Adenoassociated Virus (rAAV), thus minimizing the contamination of the rAAV production with helper virus. The objective of the study was to investigate the use of RNA interference (RNAi) directed against key adenoviral targets as an inhibitor of Ad replication. For this, lentiviral vectors encoding shRNAs for hexon, IVa2 and pol were constructed. Next, constitutive expression of the shRNAs was established in 293T cells, the parental cell line that is permissive for adenovirus replication. The shRNAs specific for hexon or IVa2 significantly promoted reduction in the level of these mRNAs as revealed by RT-qPCR quantification of the adenoviral transcripts. Next, knockdown of hexon was shown to be promising as an inhibitor of Ad replication, seen as the reduction of Ad produced in the 293T anti-hexon cell line. Both the knockdown of the hexon transcript and reduction in adenovirus replication were accentuated after cell sorting and isolation of cellular clones from the anti-hexon cell line. The anti-hexon clone showed significant reduction in the quantity of adenovirus particles when visualized by electron microscopy and 92% fewer infectious particles as compared to the parental 293T cells when full scale production was made. These results indicate that the use of shRNA technology for the inhibition of Ad replication is promising and represents the first step for the development of a strategy for the production of rAAV free from helper virus contamination

Interleukin 2 (IL-2) ist ein Zytokin, welches in menschlichen Gedächtnis-T-Helfer-Zellen (Teff Zellen) exprimiert und sekretiert wird und damit die Immunantwort formt. Im Gegensatz dazu wird IL-2 normalerweise nicht in regulatorischen T-Zellen (Treg Zellen) exprimiert, sondern von diesen nur aufgenommen. Durch die Aktivierung des T-Zellrezeptors werden Signalkaskaden induziert, welche zur Aktivierung der Transkriptionsfaktoren AP-1, NFAT, und NF-kB führen. Diese sind entscheidend für die Genexpression von IL-2. Im Rahmen meiner Dissertation habe ich die Regulation der IL-2 Genexpression untersucht. Dabei vergleiche ich die transkriptionelle Regulation in Teff Zellen mit der Regulation in Treg Zellen. Insbesondere konnte ich zeigen, dass die endogene Konzentration der Transkriptionsfaktoren sich auf die Anzahl der IL-2 Produzenten auswirkt, aber nicht auf die Konzentration von IL-2 innerhalb einer Zelle. Deshalb untersuche ich wie sich die Konzentration der Transkriptionsfaktoren auf die Häufigkeit von IL-2 Produzenten auswirkt. Ich nutze die vorhandenen endogenen Konzentrationen und kann damit vorhersagen, dass die Zahl der IL-2 Produzenten entscheidend von der c-fos Konzentration in Teff Zellen abhängt. Mit Hilfe des entwickelten Modells kann ich voraussagen, wie der spezifische Inhibitor U0126 die Häufigkeit von IL-2 Produzenten verringert. Diese Vorhersage wurde durch Experimente belegt. Meine Modelle zeigen weiterhin, dass c-fos und NFATc2 die Häufigkeit der IL-2 Produzenten in Teff Zellen kooperativ regulieren. In Treg-Zellen zeigt meine Analyse, dass alle Transkriptionsfaktoren eine ähnliche sigmoidale Wirkung auf die Häufigkeit der IL-2-Produzenten ausüben. Im Gegensatz zu den Teff Zellen haben alle Transkriptionsfaktor eine ähnliche maximale Wirkung auf Genexpression von IL-2. Mittels eines Inhibitionsmodelles konnte ich zeigen, dass der Treg zellspezifische Transkriptionsfaktor FoxP3 allen aktivierenden Transkriptionsfaktoren entgegenwirkt. / Interleukin 2 (IL-2) is a cytokine expressed in human memory T helper cells (Teff cells) and the secretion of IL-2 shapes the immune response. In contrast, human regulatory T-cells (Treg cells) commonly do not express IL-2. The gene expression of IL-2 is induced by the activation of the T-cell receptor signaling network activating the transcription factors AP-1, NFAT, and NF-kB. These transcription factors are crucial for initiating IL-2 gene expression. Within my thesis I compare the regulation of IL-2 gene expression in Teff cells and Treg cells using experiments and modeling. I demonstrate that the transcription factor concentrations correlate with number of IL-2 producers but do not affect IL-2 concentration per cell. Thus, I investigate how the transcription factor concentration of c-fos, NFATc2, p65, and p-c-jun affects the frequency of IL-2 producing cells as a proxy for the probability of a cell to produce IL-2. Using the endogenous heterogeneity of transcription factor concentrations, I predict that the number of IL-2 producers is critically dependent on the amount of c-fos in Teff cells. I use my model to predict how perturbations of c-fos by the specific inhibitor U0126 decrease the frequency of IL-2 producers in Teff cells. This prediction was than validated by experiments. My models furthermore indicate the cooperative behavior of c-fos and NFATc2 on the level of frequency of IL-2 producers in Teff cells. In Treg cells, I show that all transcription factors exert a similar sigmoidal effect on the frequency of IL-2 producers. In contrast to the effects seen in Teff cells, all transcription factor have a similar maximal effect on the IL-2 gene expression. With an inhibitory model I explore the relation between the Treg cell-specific transcription factor FoxP3 the transcription factors c-fos, NFATc2, p65, and p-c-jun on the frequency of IL-2 producers. This model indicates that FoxP3 counteracts the activating function of NFATc2, AP-1, and also NF-kB.