Submitted by JOSIANE SANTOS DE OLIVEIRA (josianeso) on 2017-05-23T17:09:12Z No. of bitstreams: 1 Felipe Rocha Wagner_.pdf: 2982994 bytes, checksum: 24c9aa0417df7a1e1754e10e2db53e9a (MD5) / Made available in DSpace on 2017-05-23T17:09:12Z (GMT). No. of bitstreams: 1 Felipe Rocha Wagner_.pdf: 2982994 bytes, checksum: 24c9aa0417df7a1e1754e10e2db53e9a (MD5) Previous issue date: 2017-02-20 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / PROSUP - Programa de Suporte à Pós-Gradução de Instituições de Ensino Particulares / A compreensão da morfologia facial, seja pela visualização ou pela antropometria, é uma questão importante para o diagnóstico clínico e estudo de síndromes dismórficas. Quando não é possível ter acesso ao paciente, fotografias 2D podem ser utilizadas como um meio de visualização e análise antropométrica, ainda que limitada a um único ponto de vista por imagem e pela perda da dimensão de profundidade, o que não ocorre em imagens 3D. Este trabalho propõe um modelo de antropometria digital e análise imersiva de modelos 3D visando auxiliar a pesquisa e o diagnóstico clínico de síndromes dismórficas. Um total de 59 crianças afetadas por Zika Congênita foram analisadas. Foram coletados modelos 3D da cabeça e 13 medidas antropométricas craniofaciais de cada paciente. Para obtenção das medidas antropométricas foram utilizadas duas abordagens, o método manual tradicional com uso do paquímetro e o método computacional proposto neste trabalho. A diferença média das medidas do método proposto em relação com o método tradicional ficou abaixo de 1mm para a maioria das medidas consideradas, sendo a maior diferença média próxima de 3mm. Na comparação de ambos, o método proposto apresentou resultados compatíveis com o tradicional além de apresentar algumas vantagens em relação à antropometria manual. / The understanding of facial morphology, either by visualization or by anthropometry, is an important issue for the clinical diagnosis and the study of dysmorphic syndromes. When it is not possible to have access to the patient, 2D photographs can be used as a means of visualization and anthropometric analysis, although limited to a single point of view per image and the loss of the depth dimension, which does not occur in 3D images. This work proposes a model of digital anthropometry and immersive analysis of 3D models aiming to aid the research and clinical diagnosis of dysmorphic syndromes. A total of 59 children affected by Congenital Zika were analyzed. 3D models of the head and 13 craniofacial anthropometric measurements of each patient were collected using two approaches, the traditional manual method with the use of the caliper and the computational method proposed in this study. The average difference of the measurements of the proposed method in relation to the traditional method was below 1mm for most of the measures considered, with the largest average difference being close to 3mm. When comparing both, the proposed method presented results compatible with the traditional one also presenting some advantages over manual anthropometry.

Les maladies virales à transmission vectorielle émergentes et ré-émergentes comme la dengue, le chikungunya ou le zika sont responsables de nombreuses épidémies sévères à travers le monde. Récemment, la propagation rapide et très étendue du virus zika (ZIKV) ainsi que les complications neurologiques graves liées à l’infection par ZIKV ont incité l’Organisation Mondiale de la Santé (OMS) à déclarer le ZIKV comme une urgence de santé publique mondiale. Aujourd’hui, les mesures préventives ou curatives de l’infection par ZIKV sont quasiment inexistantes. D’autre part, la flore endémique de La Réunion est connue comme une source riche, renouvelable et prometteuse en produits naturels anti-infectieux. L’inscription à la pharmacopée française de 19 plantes médicinales réunionnaises souligne le potentiel thérapeutique des substances naturelles issues de la biodiversité locale. Les travaux ont été consacrés à l’identification de substances naturelles anti-ZIKV issues d’une sélection de sept plantes médicinales réunionnaises inscrites à la pharmacopée française. Dans une première étape, une extraction sans solvant assistée par micro-ondes a été appliquée sur les sept plantes médicinales sélectionnées afin d’obtenir des éco-extraits. Dans une deuxième étape, le criblage de l’activité antivirale, en utilisant un clone moléculaire du ZIKV avec un gène rapporteur, a permis d’identifier deux éco-extraits actifs provenant de Aphloia theiformis et de Psiloxylon mauritianum. Après avoir vérifié l’absence de cytotoxicité et de génotoxicité des extraits actifs, l’activité antivirale a été aussi démontrée sur d’autres types d’extraits réalisés via des méthodes d’extractions traditionnelles (infusion, décoction et macération). L’activité antivirale a été validée sur différentes souches de ZIKV (historique et épidémique) ainsi que sur les 4 sérotypes du virus de la dengue. Enfin, le mode d’action des deux extraits actifs a été étudié. Il a été montré que les éco-extraits d’A. theiformis et de P. mauritianum ciblent les phases précoces du cycle viral en inhibant l’attachement du virus à la cellule hôte. À l’aide de la microscopie électronique, il a été montré que l’éco-extrait d’A. theiformis déforme la particule virale empêchant cette dernière de s’attacher à la membrane de la cellule hôte. Ces résultats démontrent l’importance des plantes médicinales réunionnaises comme source de substances naturelles anti-infectieuses. / Emerging and re-emerging vector-borne viral diseases such as dengue, chikungunya or zika are responsible for many severe epidemics worldwide. Recently, the rapid spread of zika virus (ZIKV) worldwide and the serious neurological complications associated with ZIKV infection have prompted the World Health Organization (WHO) to declare ZIKV a public health emergency. Today, preventive or curative measures against ZIKV infection are almost non-existing. On the other hand, the endemic flora of Reunion Island is known as a rich, renewable and promising source of natural anti-infective products. The registration of 19 medicinal plants from Reunion Island in the French pharmacopoeia highlights the therapeutic potential of natural substances derived from local biodiversity. The work was dedicated to the identification of natural anti-ZIKV substances from a selection of seven medicinal plants from La Reunion registered in the French pharmacopoeia. In a first step, in collaboration with a local start-up (Bourbon Extracts), a solvent-free microwave-assisted extraction was applied to the seven selected plants in order to obtain eco-extracts. In a second step, the screening for antiviral activity, using a ZIKV molecular clone with a reporter gene, allowed the identification of two candidate plants: Aphloia theiformis and Psiloxylon mauritianum. After verifying the absence of cytotoxicity and genotoxicity of the active extracts, the antiviral activity was also demonstrated on other types of extracts using traditional extraction methods (infusion, decoction and maceration). The antiviral activity has been validated on different strains of ZIKV (historical and epidemic) as well as on the four serotypes of the dengue virus. Finally, the mode of action of the two active extracts has been studied. It has been shown that the eco-extracts from A. theiformis and P. mauritianum target the early steps of the viral cycle by inhibiting the attachment of the virus to the host cell. Using electron microscopy, it has been shown that the eco-extract of A. theiformis deforms the viral particle preventing its attachment to the membrane of the host cell. These results demonstrate the importance of medicinal plants from Reunion Island as a source of natural anti-infectious substances.

En vue de contribuer à une meilleure compréhension des atteintes du système musculo-squelettique consécutives à une infection par un arthropod-borne-virus (arbovirus), deux arbovirus ont été étudiés : le virus du chikungunya (CHIKV) et le virus Zika (ZIKV), respectivement de la famille des Togaviridae et des Flaviviridae. Cette étude a été réalisée selon deux axes. Le premier s’intéresse aux atteintes articulaires consécutives à l’infection par le CHIKV. Nous avons mis au point un modèle d’imagerie in vivo reposant sur l’utilisation d’un virus recombinant exprimant la NanoLuciférase. Dans ce modèle, nous démontrons une persistance du signal bioluminescent dans les articulations 34 jours après infection. Par isolement des cartilages articulaires et des cellules constitutives, nous avons pu démontrer que les chondrocytes des cartilages métatarso-phalangiens sont infectés par le CHIKV de manière persistante, suggérant un rôle de réservoir de ces cellules. Les conséquences de l’infection au niveau cellulaire ont ensuite été étudiées in vitro. En utilisant des chondrocytes primaires humains, nous avons confirmé ces observations. De plus, les chondrocytes infectés produisent de nombreuses cytokines, induisant une stimulation du catabolisme du cartilage avec en particulier la synthèse de métalloprotéinases de matrice 3 et 9. De plus, l’infection par le CHIKV provoque la mort des cellules par apoptose, comme démontré par marquage TUNEL et par mesure de l’activité des caspases. Nous avons ensuite étudié les atteintes musculaires et le tropisme cellulaire du ZIKV. Dans un modèle murin, nous avons confirmé la présence de lésions musculaires, et l’utilisation de cellules musculaires primaires humaines a montré la sensibilité des myoblastes à l’infection, les myotubes étant résistants, suggérant un tropisme du ZIKV dépendant de la différenciation cellulaire. Trois souches virales ont été testées, sans relever de différences significatives en termes de cinétique d’infection, de nombre de cellules infectées et de production virale. L’infection des myoblastes entraîne la mort de ces cellules par un mécanisme caspase-indépendant. Des observations en microscopie électronique ont mis en évidence une vacuolisation du cytoplasme suite à l’infection, caractéristique d’une mort cellulaire par paraptose. Une analyse protéomique a démontré que l’infection des myoblastes par une souche asiatique conduit à une modification du protéome s’accentuant entre 24 heures et 48 heures post-infection, avec 225 protéines modulées 24 heures après infection contre 473 après 48 heures, indiquant une activation des voies de synthèse Interferon de type I et l’inhibition des capacités de synthèse des protéines. Ces résultats permettent une meilleure compréhension des atteintes du système-musculo-squelettique par les arbovirus / Musculoskeletal lesions caused by arthropod-borne-viruses (arboviruses) are invalidating for patients and remain poorly understood. In this study, we investigated the development of these manifestations after infection with two arboviruses: chikungunya virus (CHIKV) from the Togaviridae family, and Zika virus (ZIKV) from the Flaviviridae family. The first part of the study focused on arthritis following CHIKV infection. For this purpose, we developed a reporter virus expressing NanoLuciferase and performed experimental infections in a murine model. In vivo, a strong bioluminescent signal indicated viral replication and we observed persistence of the signal in the joints up to 34 days post-infection. By isolating primary murine cells from cartilage, we demonstrated the susceptibility of chondrocytes to CHIKV infection suggesting a role of reservoir of these cells. Furthermore, we investigated the consequences of the infection using in vitro models. We showed that primary human chondrocytes are also susceptible to CHIKV infection, which leads to the production of several cytokines involved in cartilage catabolism and induces apoptosis. In the second part of the study, we studied ZIKV muscular tropism and the associated lesions. Firstly, we confirmed the development of muscular lesions in a mouse model of ZIKA. Then, using human primary muscle cells we observed the infection of myoblasts but not myotubes, suggesting a differentiation-dependent tropism. We compared three viral strains and observed no significant difference in terms of replication, number of infected cells and viral production. Myoblasts infection induced a caspase-independent cells death mechanism and electronic microscope observations revealed intense vacuolization of cytoplasm, suggesting a paraptosis-like cell death. Proteomic analysis revealed that the Asian ZIKV strain modulated protein expression of infected cells with an increased effect after 48 hours. ZIKV-infection induced an important upregulation of biological processes associated with type I interferon and an inhibition of protein synthesis in the infected cells. These results provide valuable information about the mechanisms involved in the development of musculoskeletal lesions during arboviroses

O Zika virus (ZIKV) é um flavívirus, tal qual o virus da dengue e, assim como esse, também é transmitido pelos mosquitos do gênero Aedes, especialmente o Aedes aegipty Restrito ao continente africano até 2007, o ZIKV chegou as Américas em 2013, com os primeiros registros brasileiros da infecção datando de 2014 - 2015. Em 2016, a OMS classificou o surto de ZIKV como Emergência de Saúde Mundial. O ZIKV pode acarretar na Sindrome Congênita do ZIka Vírus (SCZ) em bebês que sofreram infecção gestacional. Além da manifestação mais temida e marcante, a microcefalia, e de demais danos neurológicos, a SCZ tem sido principalmente associada com anomalias visuais e oftalmológicas. Entretanto, poucas informações se encontram disponíveis na literatura sobre o impacto da infecção por ZIKV nas funções visuais e desenvolvimento visual em crianças sem microcefalia. Pouco se conhece também do impacto da infecção em diferentes populações brasileiras, de localidades distintas. Dessa forma, o presente trabalho se propôs a preencher essa lacuna, avaliando a acuidade visual (AV) e desenvolvimento da acuidade visual em crianças expostas gestacionalmente ao ZIKV, utilizando-se o Teste dos Cartões de Acuidade de Teller (CAT). As crianças avaliadas vieram de duas populações distintas da região Sudeste do Brasil, a cidade de Jundiaí (SP) e a cidade do Rio de Janeiro (RJ). A Coorte Zika Jundiaí foi composta de 118 crianças divididas entre grupo controle saudável (ZC) com 63 crianças, grupo experimental, dividido em dois subgrupos, baseado em exame de RT- qPCR, sorologia ou avaliação clínica, o subgrupo Exposto (ZE) com 23 crianças, em que apenas as mães tiveram infecção confirmada por ZIKV e o subgrupo Infectado (ZI), com 24 crianças no qual mãe e criança tiveram infecção confirmada. Oito crianças formaram o grupo Controle Postivo (M), com crianças com microcefalia em decorrência de outras causas. A Coorte IFF Fiocruz Rio de Janeiro foi composta de 32 crianças, todas com infecção confirmada pelo ZIKV (ZI), por exame de RT-qPCR, sorologia ou avaliação clínica. Os resultados do presente trabalho mostram que a exposição gestacional ao ZIKV, na ausência de infecção, parece não ter efeito sobre a AV ou seu desenvolvivemento. No entanto, quando ocorre infecção gestacional por ZIKV, pode haver prejuízos à AV mesmo quando a criança não apresenta anomalias neurológicas e/ou oftalmológicas aparentes. Esse trabalho, entretanto, concorda com estudos anteriores que mostram uma maior chance de haver perdas de AV concomitantementes a um comprometimento cognitivo e/ou oftalmologico. As análises do subgrupo ZI de Jundiaí dão suporte a noção de que crianças gestacionalmente infectadas por ZIKV tendem a apresentar um desenvolvimento anormal (mais lento) da AV. Esse achado, se reproduzido, possuí repercussões mais gerais, dado que quaisquer danos à função visual podem indicar deficiências no sistema nervoso central. Assim sendo, tais achados tem importantes repercussões na saúde pública. Além disso, as taxas diferentes de danos à AV, bem como de incidência de microcefalia e comprometimento oftalmológico entre as populações estudadas neste trabalho, apoiam a hipótese da existência de demarcadas diferenças regionais entre as linhagens de ZIKV circulantes no país, assim como de diferenças de suscetibilidade de hospedeiros entre as populações estudadas / The Zika virus (ZIKV) is a flavivirus, such as the dengue virus, and like the dengue virus is also transmitted by the Aedes osquitoes, especially the Aedes aegipty Restricted to the African Country until 2007, the ZIKV arrived in the Americas in 2013, with the first brazillian cases of the infection dating to 2014 - 2015. In 2016, WHO classified the ZIKV spread as a World Health Emergency. The ZIKV, in gestationally exposed children, can lead to Congenital Zika Syndrome (CZS). Beyond its most marked and feared manifestation, microcephaly, and its other neurological damages, CZS is mostly associated with visual and ophthalmologic anomalies. However, little information is available on the literature about the impact of the ZIKV infection in the visual function and on the visual development in infected children without microcephaly. Little is also known about the impact of the infection in different brazillian populations from distinct areas. With that in mind, the present work intends to fill this gap in literature, evaluating the visual acuity (VA) and visual acuity development in children gestationally exposed to the ZIKV, using the Teller Acuity Cards Test (TAC). The evaluated children were from two different populations of the Southeast region of Brazil, the city of Jundiaí (SP) and the city of Rio de Janeiro (RJ). The Zika Jundiaí Cohort was composed of 118 children divided between the healthy control group (ZC) with 63 children and the experimental group, divided in two subgroups based on RT-qPCR, sorology or clinical evaluation, the Exposed subgroup (ZE) with 23 children, which only the mothers had ZIKV confirmed and the Infected subgroup (ZI), with 24 children, wich both mother and children had ZIKV confirmed infection. Eight children formed the Positive Control Group (M), composed of children with microcephaly due to other causes. The IFF Fiocruz Rio de Janeiro Cohort was composed of 32 children, all with confirmed ZIKV infection (ZI), either by RT-qPCR/sorology exam or by clinical evaluation. The results of the present work show that ZIKV exposition in absence of infection, does not seem to affect VA or its development. However when theres a gestational ZIKV infection, it may lead to VA damages even when the children does not show any apparent neurologic/ophtalmologic anomalies. This work, nonetheless, is in agreement with former studies that show a higher chance of VA losses when the children also have cognitive and/or ophtalmologic deficits. The Jundiaí ZI Subgroup analisies support the notion that ZIKV gestationally infected children tend to have an abnormal (slower) VA development. This finding, if replicated, has more general repercussions given that any visual function damages may indicate central nervous system deficiencies. Thus, these findings have important repercussions in the public health area. Furthermore, the different rates of VA damages as well as the different rates of microcephaly incidencies and ophthalmologic deficits between the populations studied in this work support the hypothesis of marked regional differences between the brazillian ZIKV strains, as well as host suscepitibilitiess differences between the studied populations

Depuis la fin du 19ème siècle, les épidémies dues aux arbovirus constituent un problème de santé publique majeur en Nouvelle-Calédonie et dans la région Pacifique. Ces cinq dernières années, la fréquence des épidémies de dengue a augmenté et l’introduction des virus du chikungunya et du Zika (ZIKAV) a démontré que cette région du monde pouvait être un hot spot d’émergence des arboviroses. Ces trois virus sont transmis par des moustiques du genre Aedes dont le principal vecteur de la région Pacifique est Aedes aegypti. Mis à part des données sur l’épidémiologie de la dengue dans la région, peu d’informations étaient alors disponibles sur les vecteurs ou sur leurs capacités à transmettre les arbovirus. Or ces données sont cruciales pour mieux comprendre l’incidence particulière de ces épidémies dans la région.Dans ce travail de thèse, nous avons tout d’abord caractérisé génétiquement le vecteur Ae. aegypti de la région Pacifique et montré une différence génétique modérée de ce vecteur à l’échelle régionale. Nous avons ensuite, pour la première fois, évalué la compétence vectorielle d’Ae. aegypti pour les virus de la dengue 1 (DENV-1) et du ZIKAV. Les résultats obtenus montrent une faible transmission du DENV-1 et des taux de transmission hétérogènes du ZIKAV en fonction des différentes lignées virales.Les interactions entre le génotype du vecteur et le génotype viral semblent donc revêtir une importance particulière dans notre contexte. Les résultats obtenus au cours de ce travail doctoral devraient permettre une meilleure évaluation du risque de survenue d’une épidémie d’arbovirose en Nouvelle-Calédonie. / Since the end of the 19th century, epidemics due to arboviruses have been a major public health problem in New Caledonia and the Pacific region. Over the past five years, the incidence of dengue epidemics has increased and the introduction of the chikungunya and Zika viruses (ZIKAV) has demonstrated that this region of the world is to be considered as a hot spot for the emergence of arboviruses. These three viruses are transmitted by mosquitoes of the genus Aedes, whose main vector in the Pacific region is Aedes aegypti. With the exception of some data concerning the dengue virus epidemiology in the region, little was known about vectors or on their ability to transmit arboviruses. These data are crucial to increase our knowledge about the specific appearance of these epidemics in the region.During my PhD, I first genetically characterized the vector Ae. aegypti from the Pacific region and demonstrated a moderate but significant genetic differentiation. We were also the first to evaluate Ae. aegypti vector competence for dengue virus 1 (DENV-1) and for different ZIKAV strains. The results have shown a low transmission efficiency for DENV-1 and heterogeneous results for the ZIKAV transmission, these differences were linked to the ZIKAV genetic lineage.The interactions between the vector genotype and the viral genotype seem to be particularly important in our context. The results obtained during this PhD work should allow a better assessment of the risk of an arbovirus outbreak in New Caledonia as well as the development of prevention strategies.

Submitted by MARCIA ROVADOSCHI (marciar@unifra.br) on 2018-08-21T13:27:45Z No. of bitstreams: 2 Dissertacao_CristianeFracaoDiefenbach.pdf: 3471815 bytes, checksum: 5340dde1fa83817a1b28da306684977c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-08-21T13:27:45Z (GMT). No. of bitstreams: 2 Dissertacao_CristianeFracaoDiefenbach.pdf: 3471815 bytes, checksum: 5340dde1fa83817a1b28da306684977c (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-21 / The Zika Virus (ZIKV) is a flavivirus, which is responsible for causing an emerging arbovirosis in Brazil what is primarily transmitted by the bite of infected mosquitoes of the genus Aedes. A much discussed transmission route lately is blood transfusion due to the large number of asymptomatic cases and the epidemic potential of ZIKV that causes epidemics of great proportions. As such, asymptomatic individuals may contaminate blood components and thereby participate in the transfusion transmission of this infection. The disease has a benign clinical picture but serious clinical consequences have been reported, such as Guillain-Barré syndrome (GBS) and fetal malformations such as microcephaly. Due to this fact, recently, the Health Ministry (HM) declared the infection by ZIKV a Public Health Emergency of National Importance (PHENI). Thus, the major challenge for blood banks is the transfusion prevention of ZIKV due to lack of licensed laboratory routine tests for blood donor screening. In the South region of Brazil there is no epidemiological study in this population. This study will allow a robust assessment of the current epidemiological situation in this region. The objective of the study is to estimate the seroprevalence and asymptomatic viremia of ZIKV in blood donors of a Hemotherapy Service in the southern region of Brazil. In this study, blood samples were collected from 500 donors in the period from December 2016 to January 2017. The biological materials were tested for the presence of ribonucleic acid (RNA) from ZIKV, by means of real-time reverse transcription polymerase chain reaction (RT-PCR) test by commercial kit (TaqPath™ Zika Virus Kit (ZIKV) and enzyme-linked immunosorbent assay (ELISA) serologically assessed for anti-ZIKV IgG using Anti-Zika Virus ELISA IgG kit (Euroimmun, Medizinische Labordiagnostika AG, Lübek, Germany). No samples showed positive results for ZIKV RNA, which shows the low risk of transfusion transmission in the region studied. However, the serologically assessed showed the detection of a properly positive sample for anti-ZIKV IgG (0.55%) and 4 samples with optical density (OD) values in the gray zone of the test. Due to the high rate of cross-reaction between the different flaviviruses all samples (including the positive) were assessed to confirmatory testing for anti-DENV IgG (Anti-Dengue Virus ELISA IgG, Euroimmun, Medizinische Labordiagnostika AG, Lübek, Germany). The positive sample for anti-ZIKV IgG was negative for anti-DENV IgG, confirming the positive result for anti-ZIKV IgG. However, 3 gray zone seroreactive samples showed very high title of anti-DENV IgG and one remained in the gray zone. Confirmatory analysis of this sample using in-house ELISA with anti-DENV IgG antibody adsorption revealed that it was negative for anti-DENV IgG. This indicates a 0.55% seroprevalence of ZIKV in the Santa Maria-RS region. Our results demonstrate that the transfusional risk of ZIKV in the South of the Country is relatively low, however, it cannot be underestimated and serological analyses of large numbers of samples are required to determine the exact seroprevalence of anti-ZIKV and anti- DENV IgG in blood donors in the region of Santa Maria. / O vírus Zika (ZIKV) é um flavivírus responsável por causar uma arbovirose emergente no Brasil que é transmitido principalmente por picada de mosquitos infectados do gênero Aedes. Uma via de transmissão muito discutida ultimamente é a transfusão de sangue devido ao grande número de casos assintomáticos e o potencial epidêmico do ZIKV que causa epidemias de grandes proporções. Nessas ocasiões, os indivíduos assintomáticos podem contaminar hemocomponentes e deste modo participar na transmissão transfusional dessa infecção. A doença tem quadro clínico benigno, porém consequências clínicas graves têm sido relatadas, tais como a síndrome de Guillain-Barré (SGB) e malformações fetais como microcefalia. Diante desse fato, recentemente, o Ministério da Saúde (MS) declarou a infecção por ZIKV como uma Emergência em Saúde Pública de Importância Nacional (ESPIN). Assim, o principal desafio para bancos de sangue é a prevenção transfusional do ZIKV devido a falta de teste de rotina laboratorial licenciado para triagem de doadores de sangue. Na região Sul do Brasil não há nenhum estudo epidemiológico nessa população. Este estudo permitirá de uma forma robusta avaliar a situação atual epidemiológica nessa região. O objetivo do estudo é estimar a soroprevalência e a viremia assintomática do ZIKV em doadores de sangue de um Serviço de Hemoterapia da região sul do Brasil. Neste estudo foram coletadas amostras sanguíneas de 500 doadores voluntários de sangue no período de dezembro de 2016 a janeiro de 2017. Os materiais biológicos foram testados pela presença do ácido ribonucleico (RNA) do ZIKV, por meio de um teste de reação em cadeia da polimerase por transcrição reversa (RT-PCR) em tempo real por kit comercial TaqPath™ Zika Virus Kit (ZIKV) e inquérito sorológico por teste de imunoabsorbância ligada a enzima (ELISA) Anti-Zika Virus ELISA IgG kit (Euroimmun, Medizinische Labordiagnostika AG, Lübek, Alemanha). Nenhuma amostra mostrou resultado positivo para o RNA do ZIKV, o que mostra o baixo risco de transmissão transfusional na região estudada. No entanto, o inquérito sorológico revelou a detecção de uma amostra devidamente positiva para anti-ZIKV IgG (0.55%) e 4 amostras com valores de densidade ótica (DO) na zona cinza do teste. Devido a alta taxa de reação cruzada entre os diferentes flavivírus todas as amostras (incluindo a positiva) foram submetidas a teste confirmatório para anti-DENV IgG (Anti-Dengue Virus ELISA IgG, Euroimmun, Medizinische Labordiagnostika AG, Lübek, Alemanha). A amostra positiva para anti-ZIKV IgG foi negativa para anti-DENV IgG, confirmando o resultado positivo para anti-ZIKV IgG. No entanto, três amostras sororeagentes na zona cinza mostraram título muito elevado de anti-DENV IgG e uma permaneceu na zona cinza. Análise confirmatória dessa amostra via utilização de ELISA in house com adsorção de anticorpo anti-DENV IgG revelou que a mesma é negativa para anti-DENV IgG. Isso demonstra uma soroprevalência de 0.55% de ZIKV na região de Santa Maria-RS. Nossos resultados demonstram que o risco transfusional do ZIKV no Sul do país é relativamente baixo, contudo, não pode ser subestimado e análises sorológicas de grande número de amostras são necessárias para determinação da soroprevalência exata do anti-ZIKV e anti-DENV IgG em doadores de sangue na região de Santa Maria.

Congenital Zika syndrome can cause a number of changes in the child, including severe brain abnormalities. Injuries to the developing central nervous system result in sensorimotor disorders with repercussions on motor performance. Physical therapy allows the acquisition of motor skills based on the child's interaction with the environment and the task. The present study aimed to analyze the role of physiotherapy in the acquisition of the motor skills of children with congenital Zika syndrome. This is a clinical trial developed at the Physiotherapy Clinic of the University Hospital of Sergipe, with a sample of 46 children with congenital Zika syndrome. Data collection was from January 2016 to December 2017, at the physiotherapeutic admission of the child and at 18 months of age. The children were stratified according to the age group in the initial evaluation. In the first moment, a form was designed to record information on the identification and clinical characteristics of mothers and children, sociodemographic characteristics of families, neurological and musculoskeletal disorders, and type of neurological lesion. The severity of the neurological lesion was classified based on the data obtained in the medical record in the transfontanel ultrasonography report, based on the Noyola Classification System. Motor skills were assessed through the Alberta Infant Motor Scale (AIMS), stratified scores by age group and subgroup of neurological injury. The children were then submitted to a physiotherapy program, with a frequency of two weekly sessions and a duration of up to 50 minutes each. The program was based on the principles of neurodevelopment therapy (Bobath), muscle stretching and passive mobilizations. When children completed 18 months of age, motor skills reassessment was performed through the AIMS. The results showed that there was an increase in the total score comparing the initial evaluation and at 18 months of age in the children who started physical therapy with age from 0 to 6 months (p <0.0001). Despite this increase, the total scores of the study sample at 18 months were significantly lower than the normative values, with a significant difference (p <0.0001). In the initial evaluation, only children older than 4 months of age presented motor impairment when compared with normative values (p <0.0001). As for most neurological lesions, the degree was moderate to severe, with a predominance of multiple calcifications, mainly cortical-subcortical, severe cortical atrophy and agenesis / hypoplasia of the corpus callosum. The severity and type of neurological lesions in children with congenital Zika syndrome suggest impairment of neuronal migration and proliferation related to motor dysfunction. However, early physiotherapy promoted a difference in the behavior of the motor skills of these children. / A síndrome da Zika congênita pode causar um conjunto de alterações na criança, dentre elas graves anomalias cerebrais. As lesões do sistema nervoso central em desenvolvimento acarretam em desordens sensoriomotoras com repercussão no desempenho motor. A fisioterapia possibilita a aquisição de habilidades motoras a partir da interação da criança com o ambiente e com a tarefa. O presente estudo teve como objetivo analisar o papel da fisioterapia na aquisição das habilidades motoras de crianças com a síndrome da Zika congênita. Trata-se de um ensaio clínico, desenvolvido no ambulatório de fisioterapia do Hospital Universitário de Sergipe, com amostra constituída por 46 crianças com a síndrome da Zika congênita. A coleta de dados ocorreu de janeiro de 2016 a dezembro de 2017, na admissão fisioterapêutica da criança e ao completar 18 meses de idade. As crianças foram estratificadas de acordo com a faixa etária na avaliação inicial. No primeiro momento foi aplicado formulário construído para registro de informações referentes a identificação e características clinicas das mães e das crianças, características sociodemográficas das famílias, alterações neurológicas e musculoesqueléticas e tipo de lesão neurológica. Classificou-se a gravidade da lesão neurológica, a partir dos dados obtidos em prontuário no laudo da ultrassonografia transfontanela, baseada no Sistema de Classificação de Noyola. Foi realizada avaliação das habilidades motoras através da Alberta Infant Motor Scale (AIMS), estratificados escores por faixa etária e por subgrupo de lesão neurológica. Em seguida, as crianças foram submetidas a um programa de fisioterapia, com frequência de duas sessões semanais e duração de até 50 minutos cada. O programa baseou-se nos princípios da terapia do neurodesenvolvimento (Bobath), alongamentos musculares e mobilizações passivas. Quando as crianças completaram 18 meses de idade, foi realizada reavaliação das habilidades motoras por meio da AIMS. Os resultados apontaram que houve aumento do escore total comparando a avaliação inicial e aos 18 meses de idade, nas crianças que iniciaram a fisioterapia com idade de 0 a 6 meses (p<0,0001). Apesar deste aumento, os escores totais da amostra do estudo aos 18 meses são bastantes inferiores aos valores normativos, com diferença significativa (p<0,0001). Na avaliação inicial, somente as crianças com idade superior a 4 meses de idade apresentaram comprometimento motor ao comparar com os valores normativos (p<0,0001). Quanto as lesões neurológicas, em sua maioria, o grau foi de moderado a grave com predomínio de calcificações múltiplas, principalmente córtico-subcortical, atrofia cortical grave e agenesia/ hipoplasia de corpo caloso. A gravidade e o tipo das lesões neurológicas nas crianças com síndrome da Zika congênita, sugerem comprometimento da migração e proliferação neuronal, relacionados com a disfunção motora. Porém, a fisioterapia precoce promoveu uma diferença no comportamento das habilidades motoras dessas crianças. / Aracaju

Le virus Chikungunya (CHIKV), arbovirus en pleine ré-émergence, a envahi rapidement de nombreuses zones géographiques du monde. La propagation mondiale de ce virus constitue une menace pour la santé humaine car il n'y a pas de vaccin ou d'agents antiviraux appropriés pour contrôler l'infection virale. La transmission du virus s’effectue lors de la piqure d’un moustique infecté du genre Aedes, qui injecte sa salive contenant le virus dans la peau de l’hôte humain. Afin de contrôler la dissémination du virus, il est primordial de développer des recherches sur l’identification de molécules antivirales et de comprendre les mécanismes moléculaires impliqués dans les interactions hôte-virus et/ou vecteur-virus-hôte. En utilisant différentes stratégies moléculaires et cellulaires, nous avons étudié le potentiel antiviral de l'Imipramine, une molécule déjà commercialisée et qui a la capacité de perturber le transport du cholestérol intracellulaire. Nous avons démontré que cette molécule est capable d'inhiber la réplication du CHIKV dans les fibroblastes cutanés humains. Nous avons mis en évidence que l'Imipramine affectait à la fois les étapes de fusion et de réplication pendant le cycle de réplication du virus. En outre, la molécule a également fortement inhibé la réplication de plusieurs Flavivirus comme le virus Zika (ZIKV), le virus du Nil occidental et le virus de la Dengue. Nous avons également déterminé le profil protéomique global des fibroblastes humains infectés par le CHIKV ou le ZIKV. Cela nous a permis de mettre en évidence les modulations significatives de plusieurs protéines stimulées par l'interféron et de protéines impliquées dans à la défense anti-virale dans les cellules infectées. Plus important encore, nos résultats montrent pour la première fois le rôle de la protéine SAMHD1 dans l'infection des fibroblastes cutanés par les arbovirus. Enfin, compte tenu des fortes interactions entre l’hôte, le vecteur et le CHIKV, l'effet de la salive du moustique Ae. Aegypti sur l'infection virale a été étudié. À notre connaissance, cette étude est la première à montrer l’importance de la salive d’Ae. aegypti sur la facilitation de l’infection du CHIKV, dans des fibroblastes cutanés, à travers la régulation des gènes impliqués dans la réponse interféron de type I. / Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus that has been spread worldwide. The dissemination of this virus is a threat to human health since there is no approved vaccine or appropriate antiviral agents to control viral infection. The global expansion of the virus is preceded by biting of infected Aedes mosquitos, which injects saliva containing the virus into the skin of the human host. Searching for effective antiviral compounds and understanding of the molecular mechanisms involved in host-virus or vector-virus-host interactions are crucial for controlling viral spread.Using different molecular and cellular strategies, we demonstrated that the FDA approved drug, imipramine, which has the capability to disturb intracellular cholesterol transport inhibits CHIKV replication in human skin fibroblasts. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. Moreover, it also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. We have also determined the global proteomic profile of Chikungunya and Zika virus infected human skin fibroblasts, and found that several interferon-stimulated proteins and antiviral response proteins are significantly up-regulated in the infected cells. More importantly, our results also provided for the first time a role of SAMHD1 in arbovirus infection of human skin fibroblasts. Finally, we demonstrated that Aedes aegypti saliva enhances CHIKV replication in human skin fibroblasts. To our knowledge, this is the first report showing the importance of Aedes aegypti saliva on promoting CHIKV infection via down regulation of the genes involving type I IFN secretion in the infected human cutaneous cells.

This Master's thesis identifies the characteristics of the Czech media in the context of the media construction of health risks - the Zika virus, focusing on the printed media. Research by T. Knířová (2011), comparing the construction of swine flu risk between the British daily newspaper The Times and the Czech daily MF Dnes, and the analysis of the avian influenza, mad cow disease and West Nile fever in The New York Times (Shih, Wijaya, Brossard, 2008) are the initial works. The thesis uses a method of quantitative content analysis extended by an interpretative text reading. The risk of viral infection is analyzed with regard to concepts of agenda-setting, framing, and issue-attention cycles.

N-glycosylated proteins that traverse the endoplasmic reticulum (ER) can make use of the calnexin cycle to attain their correct fold. The calnexin cycle modifies the N-glycan structure and allows for association of the glycoprotein with the ER lectins calnexin and calreticulin, which in turn recruit further chaperones that assist folding. Most enveloped viruses encode glycoproteins, which, upon infection of a host cell, crucially depend on the calnexin cycle to aid their folding. This includes diverse families such as Flaviviridae, Retroviridae and Orthomyxoviridae. We studied the calnexin cycle components with the ultimate aim of developing broad-spectrum antivirals. X-ray crystallography was used to structurally characterise the murine ER α-glucosidase I, which controls entry into the calnexin cycle, with a number of inhibitory antiviral iminosugars. These data reveal flexibility in the ligands' alkyl tails and may act as a basis for the discovery of enzyme specific inhibitors. UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the quality control checkpoint of the calnexin cycle whose full-length structure from the thermophilic fungus Chaetomium thermophilum was recently determined. Presented here are a higher resolution structure in addition to SAXS studies of UGGT's interaction with Sep15, a protein that enhances UGGT activity. UGGT's reaction releases into the ER lumen UDP, which is the only known small molecule inhibitor of UGGT. An ER-resident UDPase, ENTPD5, breaks down UDP into UMP. Enzymatic characterisation of ENTPD5 reveals its substrate specificities; in addition we show a paralog, ENTPD6, possesses similar activities. Presented here is work towards crystallisation of these two proteins and a test of the anti-Zika activity of ENTPD5 inhibitors. Finally, CRISPR/Cas9 knock-out cells were generated to test, in principle, whether modulation of the activity of proteins involved in the calnexin cycle could be antiviral. The data confirm that the ER glucosidases are likely the best targets of those studied.