Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.

Submitted by Rosangela Silva (rosangela.silva3@unioeste.br) on 2018-05-24T18:44:26Z No. of bitstreams: 2 Ana Cláudia Malagutti Corsato.pdf: 1319419 bytes, checksum: c4de080588607227096826021e800839 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-05-24T18:44:26Z (GMT). No. of bitstreams: 2 Ana Cláudia Malagutti Corsato.pdf: 1319419 bytes, checksum: c4de080588607227096826021e800839 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-05 / The Diatraea saccharalis is the pest responsible for a great economic loss to the cultivation of sugar cane. As well as others members of Lepidoptera order, the D. saccharalis has a fast and efficient immune innate response, characterized by primary defense barrier, cellular immune response and humoral immune response. The antimicrobial peptides (AMPs) are included in humoral immune response that show cationic and amphiphilic characteristics besides its low molecular weight, what makes them potentially therapeutic agents. In this study were analyzed proteins with low molecular weight differently expressed in the hemolymph of D. saccharalis, after the interval of six and twelve hours of humoral immune response induction with different microorganisms compared to non-challenged worms (control). The 5th instar worms were divided in groups (n= 50) and submitted to six and twelve hours of septic challenges: control (group 1), challenged by Bacillus subtilis ATCC 6623 (group 2), challenged by Escherichia coli ATCC 11229 (group 3), and challenged by Beauveria bassiana 88 strain (group 4). In each worm was inoculated a known concentration of microorganism, and after the established time, the hemolymph was collected. The low molecular weight proteins were obtained submitting each hemolymph sample to an extraction solution containing Methanol, Acetic Acid and Water. After that, the protein extracts were concentrated in columns and protein dosage was realized in 280 nm. About 500 μg of proteins were submitted to two-dimensional electrophoresis (2-DE). The spots were excised from gels, digested with Tripsin and submitted to MALDI-ToF/ToF type mass spectrometry. The identification of protein orthologs was realized using the obtained data against the data available on the MASCOT online server (Matrix science), with the database (NCBI and Swiss rot) specified for Drosophila melanogaster and an internal database of Lepidoptera. The TagIdent tool was utilized for searching proteins through their molecular weight and isoelectric point. It was possible to identify eighteen proteins, of which twelve demonstrated to be involved with immune response in D. saccharalis. The six hours septic challenge with B. subtilis was responsible for modification of expression of Peptidoglycan recognition protein (PGRP), for increase of expression of Chitin-binding protein and for induction of expression of proteins like the Attacin-A and the Serine-protease inhibitor-like. The six hours septic challenge with E. coli increased the expression of a putative defense protein. The twelve hours septic challenge using B. subtilis and E. coli were responsible for increase of expression of Lysozyme. The six hours septic challenge with B. bassiana induced of expression of a Cecropin A2 and a Drosomycin-like. On the order hand, after twelve hours of fungal challenge, there was the induction of expression of multifunctional protein Apolipophorin-3. It was concluded that the septic challenge with different microorganisms were capable of change the expression of some proteins involved in the immune response in D. saccharalis, significant for understanding this process as well as for pointing the substances with antimicrobial functions. / A Diatraea saccharalis é a praga responsável por grandes perdas econômicas à cultura da cana-de-açúcar. Assim como outros integrantes da ordem Lepidoptera, a D. saccharalis possui resposta imunológica inata rápida e eficiente, constituída por barreira primária de defesa, resposta imune celular e resposta imune humoral. Os peptídeos antimicrobianos (PAMs) são parte da resposta imune humoral que apresentam características catiônicas e anfifílicas, além de sua massa molecular baixa, o que os tornam potenciais agentes terapêuticos. Neste estudo foram analisadas proteínas com baixa massa molecular, diferencialmente expressas na hemolinfa da D. saccharalis, após os intervalos de 6 e 12 horas de indução da resposta imune humoral com diferentes microrganismos em comparação às lagartas não desafiadas (controle). As lagartas em 5º instar foram divididas em grupos (n= 50) e submetidas a desafios sépticos de 6 e 12 horas: controle (grupo 1), desafiadas por Bacillus subtilis ATCC 6623 (grupo 2), desafiadas por Escherichia coli ATCC 11229 (grupo 3) e desafiadas por Beauveria bassiana cepa 88 (grupo 4). Em cada lagarta foi inoculada uma concentração conhecida do microrganismo e, após o tempo estabelecido, a hemolinfa foi coletada. As proteínas de baixa massa molecular foram obtidas submetendo cada amostra de hemolinfa a uma solução de extração contendo Metanol, Ácido Acético e Água. Posteriormente, os extratos proteicos foram concentrados em colunas e a dosagem proteica realizada em 280 nm. Aproximadamente 500 μg de proteínas foram submetidas à eletroforese bidimensional (2-DE). Os spots foram retirados dos géis, digeridos com Tripsina, e submetidos à espectrometria de massas do tipo MALDI-ToF/ToF. A identificação das proteínas ortólogas foi realizada utilizando os dados obtidos contra os dados disponíveis no servidor online MASCOT (Matrixscience), especificando a base de dados (NCBI e Swissprot) para Drosophila melanogaster e um banco interno de Lepidoptera. A ferramenta TagIdent foi utilizada para a busca de proteínas por meio de suas massas moleculares e ponto isoelétrico. Foram identificadas dezoito proteínas, das quais doze se mostraram envolvidas com a resposta imune em D. saccharalis. O desafio séptico de 6 horas com B. subtilis foi responsável pela alteração na expressão da proteína de reconhecimento de peptideoglicano (PGRP), pelo aumento da expressão da proteína ligadora de quitina e pela indução da expressão de proteínas como a Atacina-A e a proteína inibidora de serino-protease. O desafio imunológico de 6 horas com E. coli levou ao aumento da expressão de uma provável proteína de defesa. Os desafios de 12 horas utilizando as bactérias B. subtilis e E. coli se mostraram responsáveis pelo aumento da expressão da Lisozima. O desafio de 6 horas com B. bassiana causou indução da expressão de uma Cecropina A2 e uma possível Drosomicina. Por sua vez, após o desafio fúngico de 12 horas, houve a indução da expressão da proteína multifuncional Apolipoforina-3. Concluiu-se que desafios sépticos com diferentes microrganismos foram capazes de alterar a expressão de algumas proteínas envolvidas na resposta imune em D. saccharalis, importantes para o entendimento deste processo e também para o apontamento de substâncias com funções antimicrobianas.

Le cancer représente la deuxième cause de mortalité dans les pays industrialisés. Il a été démontré que 20% des cancers sont d'origine infectieuse. Nous nous sommes intéressés à deux oncovirus HPV (virus du papillome humain) et EBV (Epstein-Barr Virus) responsable du cancer de l'utérus et de divers lymphome B réciproquement. Les événements clés pour le développement d'un cancer viro-induit sont la persistance du virus via la dérégulation des réponses immunitaires et l'induction d'une instabilité gé¬nomique via une dérégulation du cycle cellulaire. Nous avons donc cherché si EBV était capable d'altérer la réponse immunitaire innée. Nous avons montré que EBV était capable d'inhiber TLR9 un acteur clef de la réponse immunitaire innée. Comme TLR9 est inhibé dans un certain nombre de cancers, nous nous sommes demandé si ce récepteur pouvait également, avoir un rôle dans l'oncogenèse. Nous avons montré que la réexpression de TLR9 induisait un ralentissement transitoire de la prolifération cellulaire. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation du cycle cellulaire induits par E6 une oncoprotéine de HPV16. Nous avons trouvé un nouveau mécanisme d'inhibition de l'inhibiteur du cycle cellulaire, p21. HPV16E6 se lie et inhibe les fonctions de du facteur de transcription p150Sal2, ce qui induit une inhibition de p21 dans un contexte p53 indépendant / Cancer represents the second most common cause of death in industrialized countries. Epidemiological and biological studies have now conclusively proved that a variety of infectious agents constitute one of the main causes of cancer worldwide. It has been pointed out that more than 20% of cancers are from infectious origin. HPV high-risk mucosal types are associated to 98% of all cervical cancer cases. Regarding EBV, over 90% of the world’s population is infected and can give rise to malignancies such as Burkitt lymphoma or Hodgkin disease.(Young and Rickinson 2004) Keys features for oncoviruses to induce cancer are firstly to per¬sist by dampening host immune responses and to induce genomic instability in the host by altering the regulation of the cell cycle leading the infected cells to an uncontrolled proliferation. The purpose of this thesis was to find new mechanisms by which EBV and HPV can promote carcinogenesis. We have shown that EBV can alter the regulation and expression of TLRs, the key effectors molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Our study described a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation. As TLR was found deregulated in many cancers, we hypothesized that TLR9 may also a direct role in the process of cell cycle control and that loss of its expression may lead to transformation of the cell. Our overall objective here was to study the role of TLR9 in suppressing the events that initiates transformation of epithelial cells in the setting of cervical cancer (virus-associated) and in head and neck cancer (non–virus-associated). A third project dealt with the mechanism cell cycle deregulation by the oncoprotein E6 which expressed during infection with HPV16. We reported that HPV16E6 targets the cellular factor p150Sal2, which positively regulates p21 transcription. HPV16E6 associates with p150Sal2, inducing its functional inhibition by preventing its binding to cis elements on the p21 promoter. These data described a novel mechanism by which HPV16E6 induces cell cycle deregulation with a p53-independent pathway preventing G1/S arrest and allowing cellular proliferation and efficient viral DNA replication

Chlamydia trachomatis/HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum/HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR-/-) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR-/-mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18627号 / 生博第318号 / 新制||生||42(附属図書館) / 31527 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 藤田 尚志, 教授 米原 伸, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第24749号 / 生博第490号 / 新制||生||65(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 石川 冬木, 教授 高田 穣, 教授 朝長 啓造 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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M.tb, the causative agent of pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease-based death worldwide. BCG, the only clinically approved TB vaccine, has been in use for almost a century to vaccinate against TB. Despite its success in protecting against disseminated forms of TB, it is unable to provide protection against pulmonary M.tb infection. Although there have been many recent efforts to enhance or replace BCG, our lack of understanding towards host immunity against M.tb has substantially hindered this goal. One aspect of pulmonary M.tb infection that remains poorly understood is the induction of Th1 immunity, which is substantially delayed in comparison to other pulmonary infections. This allows the bacteria to establish an infectious foothold within the host and impairs the ability of the host to clear the infection. Given the importance of the innate immune response in the induction of adaptive immunity, this delay in the establishment of Th1 immunity following pulmonary M.tb infection is likely due to a defect in the early innate immune response. However, the specific roles of this immune compartment in regards to T cell activation following pulmonary M.tb infection is still not well understood. As such, the scope of this thesis is to gain an increased understanding towards the role of the innate immune compartment in the generation of Th1 responses. Such insights will allow us to develop new strategies to improve upon future and existing TB vaccine design. / Thesis / Doctor of Philosophy (PhD)

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (M.tb) is the leading cause of infectious disease-related death worldwide. The critical role of adaptive immunity in anti-TB host defence has been firmly established; thus, current efforts in developing novel vaccination strategies against TB are primarily focused on generating protective adaptive immunity at the infection site, the lungs. Innate immunity has not been a target for vaccination strategies against TB due to the belief that innate immune cells cannot exhibit memory-like characteristics which are known to be central to the long-lasting immunity created by vaccines. Also, the importance of innate immunity in anti-TB immunity has been overlooked. However, over 25% of individuals that are heavily exposed to M.tb clear infection without any detectable conventional T cell immune responses, suggesting a crucial role for innate immune cells in bacterial clearance. Interestingly, the early protection in these individuals is associated with their Bacillus Calmette-Guerin (BCG) vaccination status. Epidemiological studies have shown that BCG is capable of providing protection against numerous infections unrelated to TB in an innate-immune dependent manner. Such observations suggest that the innate immune system exhibits memory-like characteristics, capable of remembering the exposure to the vaccine and thereby responding in an augmented manner to future systemic infections. Nonetheless, it still remains unknown whether parenteral BCG immunization modulates the innate immune cells in the lung and airways, and if so, what role the trained innate immune cells play in early protection against pulmonary TB. Using a subcutaneous BCG immunization and pulmonary TB challenge murine model, we show that early protection against M.tb is independent of adaptive responses in the BCG immunized host. Our data suggest that enhanced early protection is mediated by the BCG-trained memory alveolar macrophages that we have shown to be functionally, phenotypically, metabolically, and transcriptionally altered following immunization. These novel findings suggest a significant anti-TB immune role for the innate immune memory established in the lung following parenteral BCG immunization and have important implications for the development of novel vaccination strategies against TB. / Thesis / Master of Science (MSc) / Pulmonary tuberculosis (TB) is a disease of the lung and is now one of the leading causes of human mortality worldwide. For more than eight decades, parenterally administered Bacillus Calmette–Guérin (BCG) vaccine has been globally used as the only approved vaccine against TB. Recently, it has also been observed that BCG vaccination provides protection against other diseases unrelated to TB and reduces childhood mortality in many developing countries where it is routinely administered to children shortly after birth. The mechanisms underlying the off-target protective effects of BCG vaccine remains largely under-investigated. In this project, we investigated how BCG vaccination enhances the immune system responses against TB and other unrelated infectious diseases. A better understanding of how the BCG vaccination modulates our immune system will provide us with the knowledge that will be useful in the development of more effective vaccination strategies against infectious diseases.