No description available.

Access to abstract restricted until 05/2016. / Access to thesis restricted until 05/2016. / Department of Physics and Astronomy

There is no abstract available for this dissertation. / Department of Educational Studies

The host adaptive immune response to a pathogen infection comprises both cell mediated and antibody dependent components. Antibody mediated neutralization is a key component of protection against viruses and is the primary focus of this thesis. Antibodies recognize structurally defined epitopes within the context of native proteins. These may be represented by a simple linear sequence of amino acids or a discontinuous sequence of residues brought together by the conformational constraints of the protein. Many protein epitopes recognized by antibodies have been shown to be short α-helices of 3-5 turns. However corresponding synthetic peptides of this length have no structure in water because solvent competes strongly for the hydrogen bonding amides otherwise required to hydrogen bond one another to define an α-helix. This thesis is aimed primarily at (1) synthetically constraining short peptide sequences (9-13 residues) into stable α-helices of 3-4 turns; (2) structurally characterizing such constrained α-helical structures by circular dichroism and 1D and 2D NMR spectroscopy; and (3) evaluating these helix mimetics for serum stability, immunogenicity, antigenicity as well as the biological relevance of the antibodies they induce. The overall aim was to demonstrate that constrained short peptides more effectively structurally and functionally mimic known α-helical B cell epitopes from native proteins than unconstrained short peptides of the same lengths. The primary focus of Chapter 2 was to optimize in vitro ELISA conditions and immunization protocols for potentially assessing antibody responses in mice to short peptides corresponding to segments of important dengue virus proteins (NS1 and the envelope fusion protein, E). The NS1 peptide investigated had been suggested to be an α-helical epitope, but my investigations reveal that it is more likely a turn rather than a helix. While the E protein epitope chosen was not a viable epitope for testing a helix-constraining strategy, it was evaluated as a constrained turn mimic of a viral fusion epitope. Although the constrained peptides from both proteins (NS1 and E) elicited stronger antibody responses in mice than their unconstrained analogues, they still induced relatively poor antibody levels. Interestingly, mouse antibodies raised to the constrained peptide (β-turn analogue) from NS1 protein also reacted with the native protein. To evaluate a helix-constraining strategy for short peptides (less than 15 residues) that have no helix structure in water, an epitope of the HPV E7 protein was selected for mimicry. A short peptide sequence corresponding to this B cell epitope had previously been reported to have α-helical propensity but only in trifluoroethanol-water mixtures, and my initial work showed that it had no detectable helical structure at all in water. Chapter 3 presents an example of a short helical peptide as a B cell epitope, constrained into an α-helix by a side chain to side chain lactam bridge. The constraint involved cyclizing the peptide by specifically linking together side chains of lysine and aspartic acid inserted in the sequence three amino acids apart. CD and NMR structural studies highlighted significant α-helicity in the constrained short peptide, whereas the corresponding unconstrained short peptide had no structure in water. Both unconstrained and constrained short peptide epitopes were injected into mice and antibodies raised were quantified ex vivo by peptide ELISA. The helix-constrained epitope elicited higher antibody titres than the unconstrained peptide which was relatively non-immunogenic. Importantly, antibodies raised to the constrained synthetic α-helical peptide also reacted with the native E7 protein, suggesting that the helical constraint conferred on the peptide a structure analogous to that seen in the protein. In Chapter 4 a constrained α-helical peptide corresponding to a crystallographically defined α-helical sequence in the fusion, F protein of respiratory syncitial virus (RSV) was investigated for its potential to induce an antibody response. Again, while the helix-constrained peptide clearly had α-helicity by CD and NMR studies, the unconstrained short peptide had no detectable helical structure in water. To potentially boost antibody responses, relative to those generated against the dengue virus peptides examined in Chapter 3, both unconstrained and constrained peptides were coupled to the carrier protein KLH before immunizing mice. Significant levels of peptide reactive antibody were generated to both the unconstrained and constrained peptides. However, when investigated in a viral neutralization assay, the antibodies raised to the unconstrained peptide showed a higher neutralization potential than those raised to the constrained peptide. We attribute this unexpected difference to the fact that the region of the F protein corresponding to the epitope chosen, undergoes dramatic conformational changes during the viral fusion process and it is only in its post-fusion form that this helix has been observed. It is possible that the inherent flexibility of the linear, unconstrained counterpart of this epitope may more effectively mimic the conformational intermediates of the native structure on presentation to the immune system. Chapter 5 began an examination of the effects of three different adjuvants on antibody induction by short peptides. They were compared using a candidate peptide vaccine for malaria as a model system. As before, a helix-constrained peptide was compared with its unconstrained peptide sequence in immunization experiments. Higher titres of antibodies were raised to the constrained versus unconstrained peptides. In the second part of this chapter, a putative cancer vaccine peptide was similarly constrained via an ester linkage or a helix-inducing lactam bridge but both methods induced only low T-cell responses compared to their corresponding unconstrained sequences, possibly because the incorrect structure had been stabilized. The focus of this thesis was to evaluate a helix stabilization strategy for its possible application to short peptide vaccines. Using extensive circular dichroism and NMR spectroscopy measurements, we have shown in all cases that helix-constrained peptides were much more α-helical in solution than their corresponding unconstrained short peptide sequences that tended to have no or negligible α-helix structure in water. In some examples, we have compared serum stability and found that constrained peptides have higher serum stability than unconstrained peptides, a difference attributed to their greater stability towards proteolytic degradation – proteases being unable to recognize helices. We have also proven that the helix-constrained peptides induced higher mouse antibody titres than unconstrained peptides. Several attempts were made to boost antibody responses to the peptides by varying either immunization protocols, adjuvant or by attaching a carrier molecule. Further work is needed to optimize this promising new approach to short peptide vaccines.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 60-77).

Made available in DSpace on 2014-06-11T19:28:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-08-05Bitstream added on 2014-06-13T20:37:34Z : No. of bitstreams: 1 bachega_tf_me_jabo.pdf: 1220477 bytes, checksum: 87704e890386522830320809624f7570 (MD5) / Funep / O presente trabalho objetivou avaliar a lixiviação dos herbicidas sulfentrazone e amicarbazone aplicados a campo, com e sem óleo mineral, e correlacioná-la com o controle de corda-de-viola (Ipomoea nil e I. hederifolia), oriundas de sementes depositadas em diferentes profundidades e sob diferentes camadas de palha de cana-de-açúcar. Em área de plantio de canade- açúcar, após acumuladas precipitações de 35, 67 e 106 mm, tubos de PVC de 10 cm de diâmetro, seccionados longitudinalmente, foram enterrados até a profundidade de 35 cm. Os tubos foram retirados e, depois da última amostragem (106 mm), foram semeadas as plantas testes sorgo e Ipomoea nil em toda a seção dos tubos. Foram realizadas avaliações visuais de intoxicação aos 7, 10 e 15 dias após a semeadura (DAS) e aos 20 DAS procedeu-se a determinação da matéria seca das plântulas. O delineamento experimental utilizado foi o de blocos ao acaso, em esquema de parcelas subdivididas com quatro repetições. As parcelas consistiram da aplicação de dois herbicidas (amicarbazone e sulfentrazone), adicionados ou não de óleo, e uma testemunha sem herbicida. Nas subparcelas estudou-se as profundidades de lixiviação (0,0-2,5; 2,5-5,0; 5,0-10; 10-15; 15-20; 20-25; 25-30; 30-35 cm). Pelo bioensaio, a presença do sulfentrazone foi estimada na camada superior até os 10 cm de profundidade, mesmo com 106 mm de precipitação e independentemente da adição do óleo. / This study aimed to evaluate the lixiviation of sulfentrazone and amicarbazone applied to the field, with and without mineral oil, and its relationship with the control of morning glory (Ipomoea nil and I. hederifolia), from seed deposited at different depths and under different layers of sugar cane straw. In sugar cane field, after accumulated rainfall of 35, 67 and 106 mm, PVC pipes of 10 cm in diameter, sliced lengthwise, were buried by the depth of 35 cm. The tubes were removed and, after the last sampling (106 mm), the tests plants sorghum and Ipomoea nil were sown across the section of pipe. Visual assessments of intoxication were made at 7, 10 and 15 days after sowing (DAS) and 20 DAS the dry weight of the seedlings was determined. The experimental design was a randomized block in split plots scheme with four replications. The plot consisted of application of two herbicides (amicarbazone and sulfentrazone), with or without mineral oil, and a control without herbicide. In subplots was studied the depths of leaching (0,0-2,5; 2,5-5,0; 5,0-10, 10-15, 15-20, 20-25; 25-30; 30 - 35 cm). For the bioassay, the presence of sulfentrazone was estimated in the upper layer to the 10 cm deep, even with 106 mm of rainfall and whether adding the oil.

Resumo: O presente trabalho objetivou avaliar a lixiviação dos herbicidas sulfentrazone e amicarbazone aplicados a campo, com e sem óleo mineral, e correlacioná-la com o controle de corda-de-viola (Ipomoea nil e I. hederifolia), oriundas de sementes depositadas em diferentes profundidades e sob diferentes camadas de palha de cana-de-açúcar. Em área de plantio de canade- açúcar, após acumuladas precipitações de 35, 67 e 106 mm, tubos de PVC de 10 cm de diâmetro, seccionados longitudinalmente, foram enterrados até a profundidade de 35 cm. Os tubos foram retirados e, depois da última amostragem (106 mm), foram semeadas as plantas testes sorgo e Ipomoea nil em toda a seção dos tubos. Foram realizadas avaliações visuais de intoxicação aos 7, 10 e 15 dias após a semeadura (DAS) e aos 20 DAS procedeu-se a determinação da matéria seca das plântulas. O delineamento experimental utilizado foi o de blocos ao acaso, em esquema de parcelas subdivididas com quatro repetições. As parcelas consistiram da aplicação de dois herbicidas (amicarbazone e sulfentrazone), adicionados ou não de óleo, e uma testemunha sem herbicida. Nas subparcelas estudou-se as profundidades de lixiviação (0,0-2,5; 2,5-5,0; 5,0-10; 10-15; 15-20; 20-25; 25-30; 30-35 cm). Pelo bioensaio, a presença do sulfentrazone foi estimada na camada superior até os 10 cm de profundidade, mesmo com 106 mm de precipitação e independentemente da adição do óleo. / Abstract: This study aimed to evaluate the lixiviation of sulfentrazone and amicarbazone applied to the field, with and without mineral oil, and its relationship with the control of morning glory (Ipomoea nil and I. hederifolia), from seed deposited at different depths and under different layers of sugar cane straw. In sugar cane field, after accumulated rainfall of 35, 67 and 106 mm, PVC pipes of 10 cm in diameter, sliced lengthwise, were buried by the depth of 35 cm. The tubes were removed and, after the last sampling (106 mm), the tests plants sorghum and Ipomoea nil were sown across the section of pipe. Visual assessments of intoxication were made at 7, 10 and 15 days after sowing (DAS) and 20 DAS the dry weight of the seedlings was determined. The experimental design was a randomized block in split plots scheme with four replications. The plot consisted of application of two herbicides (amicarbazone and sulfentrazone), with or without mineral oil, and a control without herbicide. In subplots was studied the depths of leaching (0,0-2,5; 2,5-5,0; 5,0-10, 10-15, 15-20, 20-25; 25-30; 30 - 35 cm). For the bioassay, the presence of sulfentrazone was estimated in the upper layer to the 10 cm deep, even with 106 mm of rainfall and whether adding the oil. / Orientador: Pedro Luís da Costa Aguiar Alves / Coorientadora: Maria do Carmo M. Damasceno Pavani / Banca: Marcelo da Costa Ferreira / Banca: Dagoberto Martins / Mestre

abstract: The properties of adjuvants to stimulate an immune response to treat cancer has sparked a major area of research in the field of immunotherapy. Given the presence of multiple RNA sensors in mammalian host cells for eliciting innate immunity, synthetic RNA nanostructures present a unique opportunity for adjuvant exploration. While RNA nanostructures are organic and biocompatible in nature than other adjuvants, they could be tailored to have desired structural stability and functional diversity for in vivo application. In this study, a rectangular RNA origami nanostructure was designed to contain double-stranded RNA motifs and possess high structural stability. Using in vitro assays, RNA origami was shown to stimulate the toll-like receptor 3 (TLR3) signaling pathway, which has been reported to activate antigen presenting cells (APCs), natural killer (NK) cells, cluster of differentiation 8 (CD8) T-cells, and the secretion of proinflammatory cytokines. To explore RNA origami as an adjuvant for cancer immunotherapy, intraperitoneal administration of a murine colon cancer cell line (CT26) was used as a model system to mimic peritoneal metastasis (PM), in which RNA origami was investigated for its activities in mitigating PM tumor microenvironment and improving anti-tumor immunity. Given the poor outcome of the patients with PM and urgent need for new interventions, this study aims to translate the adjuvant activities of RNA origami demonstrated in vitro into potent anti-cancer immunotherapeutics. Here, it was shown that multiple intraperitoneal injections of RNA origami could inhibit tumor growth, leading to a significant delay and/or regression of metastatic tumor growth in the peritoneum. Furthermore, tumor-free mice, after being treated with RNA origami, were also resistant to a second challenge of tumor cells, indicating the development of the adaptive anti-tumor immunity. This immunity is dependent on T-cells since nude mice succumbed to tumor growth with or without RNA origami treatment. Thus, RNA-origami can function as an adjuvant to activate the innate immunity and subsequently the adaptive anti-tumor immunity, leading to tumor regression. Conceivably, RNA origami could be explored as an immunotherapeutic agent to improve the disease outcome of patients with peritoneal metastasis and peritoneal carcinogenesis. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

Le champ de la pharmacogénétique est d'une importance cruciale en oncologie pour optimiser la sélection du traitement à utiliser en fonction du profil génomique du patient et de la tumeur. En effet, au-delà des caractéristiques spécifiques de la tumeur, le génome de l'individu explique une grande partie de la variation de la réponse du observée à des agents chimiothérapeutiques à la fois en terme d'efficacité et de toxicité. Les patients atteints de cancer colorectal (CCR) sont susceptibles de recevoir une ou plusieurs lignes de chimiothérapie avec une efficacité variable et de faire l'expérience des effets secondaires connexes. Il est donc essentiel d'optimiser l’arsenal thérapeutique pour améliorer l’efficacité des traitements en évitant au maximum les effets indésirables. Le but des études de pharmacogénétique est d'étudier spécifiquement pour chaque médicament les voies métaboliques impliquées et leurs variations interindividuelles potentielles secondaires à des mutations génomiques ou somatiques. Cette recherche vise ainsi à identifier les biomarqueurs pronostiques et prédictifs qui aideront à une meilleure sélection de traitement. Pour les patients atteints de CRC, le bénéfice de survie de l'administration de la chimiothérapie adjuvante chez les patients de stade II et III reste à évaluer. En effet, l'avantage de survie donné par la perfusion de 5-fluorouracile en adjuvant (avec ou sans oxaliplatine) a été montré pour les patients de stade III CRC mais est encore indéterminé pour les patients de stade II CRC. Par définition, les mutations somatiques sont détectées dans le génome des cellules tumorales et ont été associées à la réponse aux agents chimiothérapeutiques. En outre, plusieurs rapports ont suggéré l'importance du rôle des variations héréditaires (génome constitutionnel) pour la réponse aux médicaments et à la prévision des effets secondaires. Dans ce travail, je me suis concentrée sur la relation entre les polymorphismes et la réponse aux chimiothérapies chez les patients de stade II - III CRC. J'ai observé que la cycline D1 (CCND1), les canaux sodique voltage-dépendants (SCN1A), les régulateurs de la voie WNT / β - caténine, KSR et les gènes de cellules souches cancéreuses pouvaient prédire la réponse et la survie de patients traités pour CCR en situation adjuvante. / The pharmacogenetics field is of crucial importance in oncology to optimize the selection of which chemotherapy regimen to use according to the patient’s and tumor’s genomic profile. Indeed, beyond the specific tumor characteristics, the individual’s inherited genome accounts for a large proportion of the variation in patient’s response to chemotherapeutic agents both in term of efficiency and toxicity. Patients with colorectal cancer are likely to receive one or several lines of chemotherapy with variable efficacy and to experience some related side effects. It is therefore critical to tailor the best therapeutic arsenal to improve treatments efficacy meanwhile avoiding adverse reactions susceptible to lead to treatment disruption as much as possible. The purpose of pharmacogenetics studies is to specifically investigate for each drug the implicated metabolic pathways and their potential individual variations related to genomic or somatic mutations. This research aims at identifying both prognostic and predictive biomarkers that will help for the best treatment selection. In CRC, one important issue remains to evaluate the survival benefit of adjuvant chemotherapy administration in patients with stage II and III CRC. In this setting, the survival advantage given by adjuvant 5-fluoruracil-infusion (with or without oxaliplatin) has been shown for patients with stage III CRC but is still undetermined for patients with stage II CRC. By definition somatic mutations can be found in tumor cells genome and have been related with response to chemotherapeutic agents. In addition, several reports suggested the important role of inherited variations (constitutional or germ line) for drug response and side effects prediction.