[pt] A interação social é fator fundamental para o desenvolvimento do ser humano, a partir do uso de instrumentos e de signos, sendo o processo de comunicação essencial para esse desenvolvimento. Compreendemos que a comunicação se desenvolve ao longo da vida do sujeito e é estruturada como um complexo processo de aquisição da linguagem, tendo esta uma dupla função comunicativa, a primeira como um meio de comunicação e a segunda como um processo de compreensão e representação do pensamento. Nos casos de comprometimentos severos ou déficits na comunicação, ao menos um dos envolvidos acaba sendo prejudicado. Nestes casos a utilização de comunicação alternativa vem contribuindo com subsídios para complementar, suplementar ou construir esse processo de comunicação. Sendo assim, o objetivo geral desta tese foi desenvolver um sistema de comunicação alternativa de forma colaborativa, junto às famílias de crianças com deficiência múltipla em decorrência da Síndrome Congênita do Zika Vírus (SCZV) não oralizadas com foco em atividades que estimulem o desenvolvimento da capacidade de comunicação. Esse trabalho está vinculado aos estudos realizados no Grupo de Pesquisa Educação e Mídia, Grupem, no Programa de Pós-Graduação em Educação da Pontifícia Universidade Católica do Rio de Janeiro (PUC-Rio), com o Projeto “Estudo longitudinal de intervenção e acompanhamento do desenvolvimento da habilidade de comunicação de crianças com Síndrome Congênita do Zika Vírus”, que é um subprojeto do Projeto Pesquisas e ações intersetoriais entre educação e saúde na promoção da escolarização e do desenvolvimento de crianças com Síndrome Congênita do Zika Vírus na Baixada Fluminense. A pesquisa foi realizada na região da Baixada Fluminense, RJ, com seis famílias de crianças que nasceram com a Síndrome Congênita do Zika Vírus (SCZV). Realizamos uma pesquisa qualitativa, baseada nos pressupostos metodológicos da pesquisa colaborativa. Através desta metodologia, construímos, em conjunto com as famílias das crianças participantes, alternativas para as dificuldades de comunicação apresentadas por elas, assim como para o desenvolvimento do App Comunika, um protocolo digital voltado ao desenvolvimento da capacidade de comunicação e de interação social de crianças com SCZV. O trabalho de campo foi desenvolvido em dois momentos. No primeiro realizamos visitas às casas das famílias participantes da pesquisa para realização de entrevista e aplicação de questionário socioeconômico e do Participation and Environment-Children and Youth (PEM-CY), instrumento de avaliação da capacidade de participação das crianças na família, na escola e na comunidade. No segundo momento as famílias participaram de reuniões no Instituto Multidisciplinar (IM), campus da Universidade Federal Rural do Rio de Janeiro (UFRRJ), onde iniciamos o desenvolvimento do App Comunika. Realizamos a produção de dados utilizando a observação (com registro em diário de campo), entrevistas semiestruturadas, questionário socioeconômico e o documento PEM-CY. Como referencial teórico, empregamos a teoria histórico-cultural de Vigotski, tomando seus conceitos como aportes para a compreensão das especificidades do desenvolvimento das crianças participantes e para a criação das atividades que integraram o App. A partir os dados produzidos, elaboramos as atividades que o constituem. Foram criadas 100 atividades para trabalhar a comunicação e ampliar a participação dessas crianças com base nas expectativas e necessidades apresentadas pelas famílias. De toda essa investigação emergiram como principais resultados: compreender as possibilidades advindas dos recursos de tecnologias assistivas (TA) e comunicação alternativa (CA) e abordá-las como instrumentos para a compensação de limitações em benefício dos processos de participação e comunicação das crianças com deficiência múltipla não oralizadas em decorrência da SCZV; a importância do trabalho em colaboração com as famílias para a elaboração do App, assim como da mediação na relação com o contexto, mediatizados por instrumentos, signos e símbolos desenvolvidos culturalmente; o (re) conhecimento das famílias das capacidades e diversidades de comunicação das crianças para possíveis aprendizagens futuras, assim como o quanto consideram o recurso de comunicação alternativa como uma possibilidade para trabalhar o desenvolvimento e ampliação da comunicação com seus filhos em suas casas e, posteriormente fomentando sua participação em outros contextos e com outros interlocutores; o desenvolvimento e a disponibilização do App Comunika como um recurso disponível gratuitamente na web para celular, smartphone e tablete com sistema operacional Android. / [en] Social interaction is a key factor for human development, from the use of instruments and signs, with the communication process being fundamental to this development. We understand that communication develops throughout the life of the subject, and is structured as a complex process of language acquisition, which has a dual communicative function, the first as being a means of communication and the second, as a process of understanding and representing thought. In cases of severe impairment or communication deficits, at least one of those functions end up being harmed. In these cases, the use of alternative communication has contributed with subsidies to complement, supplement or build this communication process. Therefore, the general objective of this thesis was to develop an alternative communication system in a collaborative way, together with the families of non-oralized children with multiple disabilities due to Congenital Zika Virus Syndrome (SCVZ), with focus on activities that encourage the development of communication skills. This research is linked to studies carried out in the Education and Media Research Group (Grupem), in the Graduate Program in Education at the Pontifical Catholic University of Rio de Janeiro (PUC - Rio), with the project Longitudinal study of intervention and monitoring of the development of communication skills in children with Congenital Zika Virus Syndrome, which is a subject of the project Research and intersectoral action between education and health in promoting schooling schooling and development of children with congenital zika virus syndrome at Baixada Fluminense. The research was carried out in Baixada Fluminense, RJ, with six families of children who were born with Congenital Zika Virus Syndrome (SCVZ). We carried out qualitative research, based on the methodological assumptions of collaborative research. Through this methodology, we built, with the help of the families of the participating children, alternatives for the communication difficulties presented by them, as well as the development of the app Comunika, a digital protocol aimed at developing communication skills and social interaction in children with SCVZ. The field work was carried out in two moments. On the first one, we carried out visits to the homes of the families participating in the research to carry out an interview and apply a socioeconomic questionnaire and the Participation and Environment- Children and Youth (PEM-CY), an instrument for evaluating the children s ability to participate in the family and in the community. On a second moment, the families participated in a meeting at the Multidisciplinary Institute (IM), campus of the Federal Rural University of Rio de Janeiro (UFRRJ), where we started the development of the Comunika app. We produced data using observation (with a field diary) semi-structured interviews, a socioeconomic questionnaire and the PEM-CY document. As a theoretical framework, we used Vigotski s cultural-historical theory, taking his concepts as contributions to understanding the specificities of the participating children s development, and creating the activities that integrated the app. Based on the produced data, we developed activities that made constitute it. 100 activities were created to work on communication and expand the participation of these children based on expectations and needs presented by the families. The main results emerged from all this research: understanding the possibilities arising from TA and CA resources and approaching them as instruments for compensating limitations in favor of the participation and communication processes of children with multiple non-oralised due to SCVZ; the importance of working in collaboration with families for the development of the app, as well as the meditation in the relationship with the context, mediated by culturally developed instruments, signs and symbols; the knowledge of families of children s communication skills and diversity for possible future learning, as well as how much they consider the alternative communication resource as a possibility to work on the development and expansion of communication with their children at home and, subsequently, encouraging their participation in other contexts and with other interlocutors; the development and availability of the Comunika app as a resource available free of charge on the web for cell phones, smartphones and tablets with operating Android system.

Submitted by admin tede (tede@pucgoias.edu.br) on 2017-04-27T14:46:13Z No. of bitstreams: 1 JÉSSICA BARLETTO DE SOUSA BARROS.pdf: 1338303 bytes, checksum: f224cbe0cdd0f2bfa58ecbb4e530e87a (MD5) / Made available in DSpace on 2017-04-27T14:46:13Z (GMT). No. of bitstreams: 1 JÉSSICA BARLETTO DE SOUSA BARROS.pdf: 1338303 bytes, checksum: f224cbe0cdd0f2bfa58ecbb4e530e87a (MD5) Previous issue date: 2017-03-16 / The Zika virus (ZIKV) is a Flavivirus belonging to the family Flaviviridae, which was initially found and isolated on a Rhesus monkey in the Zika forest, Africa, in 1947. It is responsible for causing Zika fever, characterized mainly by symptoms such as fever, headache, Arthralgia, myalgia, and maculopapular rash. Currently, ZIKV infection has been considered an emerging and endemic infection and therefore several clinical aspects of the disease have been clarified, but there are still inflammatory features that need to be elucidated. The aim of the present study was to evaluate the inflammatory cytokines (IL-1β, IL-2, IL-5, IL-6, IL-7, IL-9, IL-12p70, IL-15 and IL-17A), anti-inflammatory (IL-4, IL-10, IL-13 and IL-1ra), and acute phase proteins (C-reactive protein and ferritin) in ZIKV-infected patients. We observed alterations in levels of C-reactive protein in ZIKV-infected patients when compared to healthy donors, and it was verified in the acute and convalescent phase. In relation to ferritin levels, they did not present significant changes at any point in the study. An increase in IL-5, IL-7 and IL-9 was also observed when compared to the acute and control groups. No changes were found in IL-4, IL-10 and IL-13 cytokines, however significant levels of IL-1ra were detected in ZIKV-infected individuals. Regarding the acute and convalescent phase, only IL-7 presented statistically significant values. All these molecules were correlated with the amount of signs and symptoms, and no statistical difference was observed. / O Zika vírus (ZIKV) é um Flavivírus pertencente à família Flaviviridae, que foi inicialmente encontrado e isolado em um macaco Rhesus na floresta Zika, África, em 1947. É responsável por ocasionar a febre Zika, caracterizada principalmente por sintomas como febre, cefaleia, artralgia, mialgia e exantema maculopapular. Atualmente, a infecção pelo ZIKV têm sido considerada uma infecção emergente e endêmica e por isso diversos aspectos clínicos da doença já foram esclarecidos, mas ainda existem características inflamatórias que precisam ser elucidadas. Assim, o presente trabalho objetivou avaliar as citocinas inflamatórias (IL-1β, IL-2, IL-5, IL-6, IL-7, IL-9, IL-12p70, IL-15 e IL-17A), anti-inflamatórias (IL- 4, IL-10, IL- 13 e IL-1ra), e proteínas de fase aguda (proteína C reativa e ferritina) em infectados pelo ZIKV. Observamos alterações nos níveis da Proteína C Reativa em infectados pelo ZIKV quando comparados aos doadores saudáveis, o mesmo foi verificado na fase aguda e convalescente. Em relação aos níveis de ferritina, esses não apresentaram alterações consideráveis em nenhum momento do estudo. Também foi constatado um aumento da IL-5, IL-7 e IL-9, quando comparado os níveis da fase aguda e grupo controle. Não foram encontradas alterações nos níveis das citocinas IL-4, IL-10 e IL-13, no entanto níveis significativos de IL-1ra foram detectados em infectados pelo ZIKV. Em relação a fase aguda e convalescente, apenas a IL-7 apresentou valores significativamente estatísticos. Todas essas moléculas foram correlacionadas com a quantidade de sinais e sintomas, e nenhuma diferença estatística foi observada.

O Zika vírus (ZIKV) pertence à família flaviviridae que inclui espécies transmitidas principalmente por mosquitos Aedes sp. No Brasil, casos de microcefalia fetal foram associados à infecção congênita por ZIKV. Vários mecanismos de imunorregulação operantes durante a gestação como a inflamação gerada e resposta antiviral são vitais para o entendimento da infecção congênita do ZIKV. Desta forma, é proposto avaliar se a inflamação via ativação do receptor Toll-like 4 (TLR4-LPS) em vilos placentários humanos e em modelo murino interfere na infecção por ZIKV. Para isso, explantes de vilos placentários humanos de 3o trimestre foram ativados com LPS e posteriormente infectados com ZIKV. Nossos achados mostram que a ativação com LPS é capaz de aumentar a replicação de ZIKV em vilos placentários e de elevar a atividade enzimática da lactato desidrogenase, indicativo de ativação celular. A replicação viral foi inibida com o tratamento dos explantes com um antioxidante, Naringenina, mostrando potencial terapêutico. A ativação placentária com LPS inibiu a via IRF-3, diminuindo a atividade antiviral e aumentando a clivagem proteica do componente da via de autofagia LC3, sendo que estes mecanismos podem estar relacionados com o aumento da replicação viral. Em paralelo, foi detectado aumento da produção de citocinas inflamatórias e hiperplasia das células de Hofbauer nos explantes inflamados e infectados, indicando que estes macrófagos fetais podem ser um nicho de replicação viral. Em modelo murino, evidenciamos que a inoculação intravaginal de LPS em fêmeas prenhas previamente à infecção com ZIKV é capaz de promover o aumento de carga viral no cérebro das mães e da prole, com agravamento do quadro de microcefalia nos fetos. Em conjunto, os dados mostram que em modelo de explante placentário humano e, in vivo , em camundongos, a inflamação é fator predisponente da replicação do ZIKV. As estratégias imunomoduladoras mostram potencial terapêutico, atenuando a resposta inflamatória e a diminuição da replicação viral. / The Zika virus (ZIKV) belongs to the flaviviridae family that includes species transmitted mainly by mosquitoes Aedes sp. In Brazil, cases of fetal microcephaly were associated with ZIKV congenital infection. Several mechanisms of immunoregulation operative during gestation as the inflammation generated and antiviral response are vital for the understanding of congenital ZIKV infection. Thus, it is proposed to evaluate whether inflammation via Toll-like receptor 4 (TLR4-LPS) activation in human and murine placental villi interferes with ZIKV infection. For this, human placental explants of 3rd trimester were activated with LPS and later infected with ZIKV. Our findings show that LPS activation is capable of increasing ZIKV replication in placental villi and elevating the enzymatic activity of lactate dehydrogenase, indicative of cellular activation. Viral replication was inhibited with the treatment of explants with an antioxidant, Naringenin, showing therapeutic potential. LPS placental activation inhibited the IRF-3 pathway, decreasing antiviral activity and increasing protein cleavage of the autophagy pathway component LC3, and these mechanisms may be related to increased viral replication. In parallel, increased production of inflammatory cytokines and Hofbauer cell hyperplasia were detected in inflamed and infected explants, indicating that these fetal macrophages may be a niche for viral replication. In the murine model, we demonstrated that the intravaginal inoculation of LPS in pregnant females prior to infection with ZIKV is able to promote the increase of viral load in the brains of mothers and offspring, with worsening of the microcephaly in fetuses. Together, the data show that in the model of human placental explant and, in vivo, in mice, inflammation is a predisposing factor of ZIKV replication. Immunomodulatory strategies show therapeutic potential, attenuating the inflammatory response and decreasing viral replication.

Submitted by Marcio Maciel Bastos (marciomacielbastos@hotmail.com) on 2018-04-02T15:09:10Z No. of bitstreams: 4 dsrtcao.pdf: 3763696 bytes, checksum: e5614aca9d57fde04661fc6da2f7fe8d (MD5) Folha de assinaturas.pdf: 145939 bytes, checksum: b0efa18a3054171cdc8cc52a6a4d715f (MD5) EMAp Ficha catalográfica.pdf: 16727 bytes, checksum: 10446f26497b911613967664f6ca959c (MD5) dissertacao.pdf: 3462226 bytes, checksum: 2cb3f716b95b7d4ba28b55bcf90ed3c8 (MD5) / Approved for entry into archive by Janete de Oliveira Feitosa (janete.feitosa@fgv.br) on 2018-04-06T14:17:00Z (GMT) No. of bitstreams: 4 dsrtcao.pdf: 3763696 bytes, checksum: e5614aca9d57fde04661fc6da2f7fe8d (MD5) Folha de assinaturas.pdf: 145939 bytes, checksum: b0efa18a3054171cdc8cc52a6a4d715f (MD5) EMAp Ficha catalográfica.pdf: 16727 bytes, checksum: 10446f26497b911613967664f6ca959c (MD5) dissertacao.pdf: 3462226 bytes, checksum: 2cb3f716b95b7d4ba28b55bcf90ed3c8 (MD5) / Approved for entry into archive by Marcia Bacha (marcia.bacha@fgv.br) on 2018-04-12T18:20:43Z (GMT) No. of bitstreams: 4 dsrtcao.pdf: 3763696 bytes, checksum: e5614aca9d57fde04661fc6da2f7fe8d (MD5) Folha de assinaturas.pdf: 145939 bytes, checksum: b0efa18a3054171cdc8cc52a6a4d715f (MD5) EMAp Ficha catalográfica.pdf: 16727 bytes, checksum: 10446f26497b911613967664f6ca959c (MD5) dissertacao.pdf: 3462226 bytes, checksum: 2cb3f716b95b7d4ba28b55bcf90ed3c8 (MD5) / Made available in DSpace on 2018-04-12T18:21:12Z (GMT). No. of bitstreams: 4 dsrtcao.pdf: 3763696 bytes, checksum: e5614aca9d57fde04661fc6da2f7fe8d (MD5) Folha de assinaturas.pdf: 145939 bytes, checksum: b0efa18a3054171cdc8cc52a6a4d715f (MD5) EMAp Ficha catalográfica.pdf: 16727 bytes, checksum: 10446f26497b911613967664f6ca959c (MD5) dissertacao.pdf: 3462226 bytes, checksum: 2cb3f716b95b7d4ba28b55bcf90ed3c8 (MD5) Previous issue date: 2018-03-06 / The Zika virus (ZIKV) is a pathogen of the family Flaviviridae, transmitted in Brazil mainly by the mosquito Aedes aegypti and in less extent by sexual relations. In addition to symptoms common to dengue and chikungunya, the zika virus is also capable of causing irreversible damage to the nervous system, in adults it is related to Guillain-Barr´e syndrome and in fetuses it causes microcephaly. The Health Department of Rio de Janeiro maintains a database with records of patients who sought care and was infeccted with Zika. Our study seeks to estimate the true size of the epidemic that occurred in the year 2016 and the parameters that fit to explain the dissemination process. To make these estimates, we used the data provided by the Health Department and a hierarchical Bayesian model adapted to the SIR epidemiological model. We perform the inference process through modern sampling techniques such as Automatic Differentiation Variational Inference (ADVI), Stein Variational Gradient Descent (SVGD) and No-U-Turn (NUTS). / O Zika virus (ZIKV) é um patógeno da família Flaviviridae transmitido no Brasil principalmente pelo mosquito Aedes aegypti e em menor escala por relações sexuais. Além dos sintomas comuns à dengue e chikungunya, o vírus da zika também é capaz de causar danos irreversíveis no sistema nervoso, em adultos está relacionada à síndrome de Guillain-Barré e em fetos provoca microcefalia. O sistema de saúde do Rio de Janeiro mantém um banco de dados com os registros dos pacientes que buscaram atendimento e apresentaram sintomas de Zika. O nosso estudo busca estimar o verdadeiro tamanho da epidemia que ocorreu no ano de 2016 e os parâmetros que podem ser ajustados para explicar o processo de disseminação. Para realizar essas estimativas, utilizamos os dados fornecidos pelo sistema de saúde e uma modelagem Bayesiana hierárquica adaptada ao modelo epidemiológico SIR. Realizamos o processo de inferência através de modernas técnicas de amostragem, como Automatic Differentiation Variational Inference (ADVI), Stein Variational Gradient Descent (SVGD) e No-U-Turn (NUTS).

Au cours des récentes épidémies, l’infection par le virus Zika (ZIKV) est à l’origine de microcéphalies chez le fœtus, d’avortements spontanés et du syndrome de Guillain–Barré chez l’adulte. L’infection par ZIKV représente donc un problème de santé publique. Il est nécessaire de comprendre la biologie de ce virus et quelles sont les réponses cellulaires impliquées. Dans les cellules infectées, les virus sont détectés par des récepteurs de la réponse immunitaire innée, tels que les Toll-like receptors (TLR). Cette reconnaissance conduit à la production de molécules antivirales, tel que l’interféron (IFN) de type I et III. De récents travaux montrent que l’activation de la voie TLR3 contribuerait au contrôle de la propagation de ZIKV et sa pathogénèse. Nous avons mis en place une approche pour cartographier les interactions génétiques entre le génome viral et différents régulateurs et effecteurs de cette réponse antivirale, incluant les étapes de reconnaissance du virus par TLR3. Cette approche est basée sur une combinaison de biologie moléculaire et d’évolution expérimentale qui repose sur le taux élevé de mutation de ZIKV et sa rapide faculté d’adaptation. J’ai pu mettre en avant l’apparition d’un phénotype de ZIKV face aux défenses antivirales de lignées cellulaires humaines qui se caractérise par une résistance à l’activation de la voie TLR3 et une meilleure propagation du virus après 5 passages. J’ai été capable d’observer que la population virale adaptée présente une perte d’activation des réponses antivirales (i.e. ISG15, MxA) 3h post-infection. La mise au point et la validation de l’approche CirSeq permet une analyse de précision des données de séquençage haut-débit, et permettra ainsi de prédire l’évolution de mécanismes d’échappement viral. / Zika virus (ZIKV) infection causes neurological diseases and birth defects representing an important threat for human health. Recent studies demonstrated that interferon (IFN) response is pivotal for the control of ZIKV spread, its in utero transmission and protects against ZIKV-induced neurological diseases. It is necessary to understand the biology of this virus and what are the cellular responses involved. In infected cells, viruses are detected by receptors of the innate immune response, such as Toll-like receptors (TLRs). This recognition leads to the production of antiviral molecules, such as type I and III interferon (IFN). Recent work shows that activation of the TLR3 pathway contributes to controlling the spread of ZIKV and its pathogenesis. We have implemented an approach to map the interactions between the viral genome and different regulators and effectors of this antiviral response, including the TLR3 virus recognition steps. This approach is based on a combination of molecular biology and experimental evolution based on the high mutation rate of ZIKV and its rapid adaptation. I was able to highlight the appearance of a phenotype of ZIKV against the antiviral defenses of human cell lines that triggered resistance to activation of the TLR3 pathway and a better spread of the virus after 5 iterative passages. I was able to observe that the adapted viral population exhibited a loss of activation of antiviral responses (i.e. ISG15, MxA) 3h after infection. The implementation and validation of the CirSeq approach allows an analysis of the accuracy of high throughput sequencing data, as well as predicting the evolution of viral escape mechanisms.

abstract: The role of climate change, as measured in terms of changes in the climatology of geophysical variables (such as temperature and rainfall), on the global distribution and burden of vector-borne diseases (VBDs) remains a subject of considerable debate. This dissertation attempts to contribute to this debate via the use of mathematical (compartmental) modeling and statistical data analysis. In particular, the objective is to find suitable values and/or ranges of the climate variables considered (typically temperature and rainfall) for maximum vector abundance and consequently, maximum transmission intensity of the disease(s) they cause. Motivated by the fact that understanding the dynamics of disease vector is crucial to understanding the transmission and control of the VBDs they cause, a novel weather-driven deterministic model for the population biology of the mosquito is formulated and rigorously analyzed. Numerical simulations, using relevant weather and entomological data for Anopheles mosquito (the vector for malaria), show that maximum mosquito abundance occurs when temperature and rainfall values lie in the range [20-25]C and [105-115] mm, respectively. The Anopheles mosquito ecology model is extended to incorporate human dynamics. The resulting weather-driven malaria transmission model, which includes many of the key aspects of malaria (such as disease transmission by asymptomatically-infectious humans, and enhanced malaria immunity due to repeated exposure), was rigorously analyzed. The model which also incorporates the effect of diurnal temperature range (DTR) on malaria transmission dynamics shows that increasing DTR shifts the peak temperature value for malaria transmission from 29C (when DTR is 0C) to about 25C (when DTR is 15C). Finally, the malaria model is adapted and used to study the transmission dynamics of chikungunya, dengue and Zika, three diseases co-circulating in the Americas caused by the same vector (Aedes aegypti). The resulting model, which is fitted using data from Mexico, is used to assess a few hypotheses (such as those associated with the possible impact the newly-released dengue vaccine will have on Zika) and the impact of variability in climate variables on the dynamics of the three diseases. Suitable temperature and rainfall ranges for the maximum transmission intensity of the three diseases are obtained. / Dissertation/Thesis / Doctoral Dissertation Applied Mathematics 2018

abstract: Zika virus (ZIKV) outbreaks have been linked to several neurological pathologies in the developing fetus, which can progress to spontaneous abortion and microcephaly in newborns whose mothers were infected with the virus during pregnancy. ZIKV has also been correlated with neurological complications in adults such as Guillain-Barré Syndrome (GBS). ZIKV outbreaks often occur in low income areas with limited access to healthcare. Therefore, there is a need to create a low-cost preventative vaccine against the virus. Mature ZIKV particles contain a lipid bilayer, a positive sense single stranded RNA genome and three structural proteins: the envelope (E), membrane (M) and capsid (C) proteins. Congruently, to other members of the Flaviviridae family, ZIKV proteins are synthesized as a polyprotein precursor which needs to be processed to release the mature structural and non-structural viral proteins. Past studies have determined the ZIKV precursor protein is cleaved by a host furin protease which separates the Pr peptide and the M protein, while the host signal peptidase separates the M and E protein. Processing is important for correct folding of the E protein. In turn, the most important neutralizing antibodies upon infection are directed against epitopes of the E protein. In this work, we used a Bean Yellow Dwarf Viral vector system to transiently express, in Nicotiana benthamiana plants, a portion of the ZIKV polyprotein encoding the Pr, M and E proteins. I further demonstrate that plants can proteolytically process the polyprotein to yield the two integral membrane proteins M and E. These proteins can be shown to co-partition into a soluble membrane-particulate fraction, consistent with formation of enveloped virus-like particles (VLPs). This work provides the first step in creating a low-cost sustainable plant-based production system of ZIKV VLPs that can be explored as a potential component 0f a low-cost prophylactic vaccine against ZIKV. / Dissertation/Thesis / Masters Thesis Molecular and Cellular Biology 2018

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Considered viral diseases of major re-emerging in the world, the dengue, chikungunya and Zika have as the main vector Aedes aegypti (L.) (Diptera: Culicidae). Larvicidal vector control is an important measure to prevent the transmission of such infections. A major challenge in the control of this arthropod is the low sensitivity of its population through the use of conventional larvicides. Thus, the insect acquires resistance, reducing the effectiveness of these pesticides. Consequently, it will lead to an increase in the risks of toxicity in nontarget organisms and a change in the environment. An alternative to avoid problems caused by the use of these products is the search for new compounds with less environmental impact and better benefits to human health. The indole molecule ring represents one of the subunits of great importance in the discovery of new pesticide products for the pharmaceutical market. By Friedel-Crafts acylation reaction, the C-3 of this ring makes it susceptible to chemically react. Thus, 12 indole analogues were synthesized as potential larvicidal agents against Ae. aegypti in its 3rd larval stage followed by the evaluation of the toxicity in nauplii of Artemia sp. The compounds were identified by analytical thin-layer chromatography, purified on a silica gel 60 chromatographic column (using the Hexane: Ethyl acetate (90:10, v / v) binary system as the mobile phase) and characterized by melting point, 13C and 1H NMR (using a residual solvent peak or TMS as reference for 1 H NMR spectra), mass spectrum and infrared. Bioassays were performed using 20 larvae per test, disposable cups containing 20mL of the test solution in triplicate. Branched aliphatic side chain derivatives were more potent than the others were, and the linear ones exhibited potency oscillation as the addition of the methylene chains. Toxicity tests indicated that (3-chlorophenyl)1-(1H-indol-3-yl)methanone, with moderate larvicidal potency (LC50 = 50.59 ppm), showed the highest selectivity index (SI >19.7), being less toxic to Artemia sp. than Ae. aegypti. The relationships between structural changes in indole derivatives and their LC50 results provide information that may contribute to the understanding of the influence of physicochemical properties on the larvicidal action of this class of compounds, without damage to the ecosystem. / Consideradas doenças virais de grande reemergência no mundo, a dengue, chikungunya e zika têm como principal vetor o Aedes aegypti (L.) (Diptera:Culicidae). O controle vetorial baseado em larvicidas é uma medida importante de prevenir a transmissão de tais infecções. Um grande desafio nas formas de controle desse artrópode está na baixa sensibilidade de sua população através do uso de larvicidas convencionais. Assim, o inseto adquire resistência, reduzindo a eficácia desses pesticidas. Consequentemente, levará a um aumento nos riscos de toxicidade em organismos não-alvo e uma alteração ao meio ambiente. Uma alternativa para evitar problemas ocasionados pela utilização desses produtos é a pesquisa por novos compostos com menos impacto ambiental e melhores benefícios à saúde humana. O anel da molécula do indol representa uma das subunidades com grande importância na descoberta de novos produtos pesticidas para o mercado farmacêutico. Por reação de acilação de Friedel-Crafts, o C-3 deste anel torna suscetível a reagir quimicamente. Assim, foram sintetizados 12 análogos do indol como potenciais agentes larvicidas contra o Ae. aegypti no seu 3º estágio larvar, seguido pela avaliação da toxicidade em náuplios de Artemia sp. Os compostos sintetizados foram identificados por cromatografia em camada delgada analítica, purificados em coluna cromatográfica de sílica gel 60 (utilizando o sistema binário Hexano: Acetato de etila (90:10, v/v) como fase móvel), e caracterizados por ponto de fusão, RMN de 13C e 1H (utilizando um pico de solvente residual ou TMS como referência para os espectros de RMN 1H), espectro de massas e infravermelho. Os bioensaios foram realizados utilizando 20 larvas por teste, copos descartáveis contendo 20 mL da solução teste em triplicata. Derivados de cadeias laterais alifáticas ramificadas foram mais potentes que os demais, e os lineares exibiram oscilação de potência conforme acréscimo das cadeias de metileno. Ensaios de toxicidade apontaram que a (3-clorofenila)1-(1H-indol-3- ila)metanona, com potência larvicida moderada (CL50 = 50,59 ppm), exibiu o maior índice de seletividade (IS >19,7), sendo menos tóxico para Artemia sp do que para o Ae. aegypti. As relações entre mudanças estruturais dos derivados do indol e seus resultados de CL50 fornecem informações que podem contribuir para a compreensão da influência de propriedades físico-químicas na ação larvicida desta classe de compostos, sem prejuízos ao ecossistema. / São Cristóvão, SE

Les arbovirus sont un groupe de virus transmis par des vecteurs arthropodes contaminant chaque année plusieurs centaines de millions de personnes à travers le monde. De nombreux facteurs environnementaux, comme les xénobiotiques peuvent influencer la propagation des infections virales, la susceptibilité de l'hôte et la sévérité de l'infection. Parmi eux, le cadmium, métal toxique est connu pour moduler la résistance de l'hôte lors des infections par les arbovirus. Ces travaux de thèse se sont portés sur l'étude de l'alphavirus Sindbis et du flavivirus Zika infectant des cellules épithéliales humaines. Les résultats de cette thèse ont permis de démontrer que les cellules A549 étaient permissives à la souche épidémique de 2013 du virus Zika. Le virus se réplique efficacement et induit une apoptose de type mitochondriale dans les cellules A549. La réponse immunitaire innée des cellules a été caractérisée et le rôle des interférons de type I a été mis en avant. Ces résultats peuvent contribuer à une meilleure compréhension des mécanismes de pathogénicité de ce virus chez l'homme. Par la suite, les effets d'une exposition au cadmium lors des infections par les virus Sindbis et Zika ont été évalués. L'infection par le virus Zika n'a pas été modulée par l'exposition au cadmium in vitro. Mais de façon surprenante dans le cas de l'infection des cellules HEK 293, le cadmium a protégé les cellules des effets cytopathiques induits par le virus Sindbis. En présence de cadmium, l'apoptose induite par le virus a été inhibée et la réplication du virus Sindbis a été réduite. / Arboviruses are a group of viruses transmitted by arthropod vectors infecting hundreds of millions of people each year worldwide. Many environmental factors including xenobiotics can influence the spread, the susceptibility and the outcome of viral infections. Among them, cadmium, a toxic metal is known to affect the host resistance against arboviral infection. In this thesis, our work has been focused on studying the infection capability of the Sindbis alphavirus and the Zika flavivirus in human epithelial cells. The results demonstrated that A549 cells was permissive to the 2013 epidemic strain of Zika virus. The virus replicates efficiently leading to mitochondrial apoptosis. The innate immune response was characterized and the crucial role of type I interferon was highlighted. These results may contribute to a better understanding of Zika virus pathogenesis in humans. Thereafter, the effects of cadmium exposure on Sindbis virus and Zika virus infection was evaluated. Zika virus infection was not modulated by cadmium in vitro. Interestingly, cadmium protected the HEK 293 cells from the cytopathic effect induced by Sindbis virus. Cadmium exposure inhibited the apoptosis and reduced the viral replication.

<div><p>Zika virus (ZIKV) is a mosquito-borne pathogen that stayed unnoticed for over half a century. Only after the 2015-16 Brazilian outbreak did the severity of the infectious outcome, particularly the Congenital Zika Syndrome, become apparent. ZIKV is associated with severe neurodevelopmental impairments in human fetuses, including microencephaly, ventriculomegaly, retinopathy, and sensorineural hearing loss. Though the pandemic is now under control in the Latin American countries, several tropical countries could still be at risk of widespread infection. This warrants a better understanding of the congenital Zika syndrome; this project attempts to contribute towards this goal.</p><p><br></p><p>Previous reports examining neural progenitor tropism of ZIKV in organoid and animal models did not address whether the virus infects all neural progenitors uniformly. To explore this, ZIKV was injected into the neural tube of 2-day-old chicken embryos, resulting in non-uniform periventricular infection 3 days later. Recurrent foci of intense infection were present at specific signaling centers that influence neuroepithelial patterning at a distance through secretion of morphogens. ZIKV infection reduced transcript levels for 3 morphogens, SHH, BMP7, and FGF8, expressed at the midbrain basal plate, hypothalamic floor plate, and isthmus, respectively. Levels of Patched1, a SHH-pathway downstream gene, were also reduced and a SHH-dependent cell population in the ventral midbrain was shifted in position. Thus, the diminishment of signaling centers through ZIKV-mediated apoptosis may yield broader, non-cell autonomous changes in brain patterning.</p><p><br></p></div><p>Sensorineural hearing loss is a relatively understudied consequence of congenital Zika syndrome, and balance disorders are essentially unreported to date. ZIKV pathogenesis was explored in the developing inner ear using the accessible chicken embryo model system. One goal was to assess the spatiotemporal susceptibility of otic epithelial-derived structures to ZIKV infectivity. Direct injections of the inner ear or the inner ear primordium were performed <i>in ovo</i>with subsequent harvests at 2 to 8 days-post-infection. The degree of infection in sensory/prosensory organs was evaluated histologically to determine the susceptibility of one auditory and five vestibular organs. ZIKV infection of the sensory as well as non-sensory epithelia was observed at most stages of analysis, with no apparent preference for one over the other. The lagena, the ventral most tip of the chicken inner ear, and the endolymphatic sac/duct were least frequently infected. In this report, two novel findings in sequela of ZIKV infection are presented: the vestibular labyrinth can present with stalled canal morphogenesis, and the auditory ganglion can be severely shrunken, perhaps due to an increased cell death upon early ZIKV infection of the inner ear.</p><p><br></p><p>Additional methods of peripheral infection in the chicken embryos were tested to examine ZIKV transmission to the central nervous system: E3 blood vessel, E4 limb bud, and E10 chorioallantoic membrane infections. Although none of these methods resulted in a histologically significant infection of the developing brain 3 to 6 days-post-infection, evidence of ZIKV genome replication and viremia was detected in several tissue types.<br></p>