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Orientador: Flávia Hebeler Barbosa Trovão / Resumo: Bibliotecas conformacionais de anticorpos vem constituindo importantes ferramentas na investigação científica de imunoglobulinas expressas em diferentes situações biológicas incluindo patologias. O conhecimento e elucidação estrutural de anticorpos diferenciais produzidos em patologias distintas pode representar ferramenta molecular fundamental para a intervenção na fisiopatologia da doença; incluindo diagnóstico diferencial, identificação de biomarcadores, terapêutica baseada em imunoglobulinas para bloqueio de patógenos bem como, contribuir para a constituição de painéis de anticorpos capazes de identificar epítopos agregando, também, informações para avanços no desenvolvimento de vacinas. A maior dificuldade atualmente reportada na literatura científica é a obtenção in vitro dos fragmentos Fab (Fragment Antigen Binding) devido à ausência de informações metodológicas das fases experimentais necessárias à construção do Fab, substrato essencial a construção de bibliotecas de anticorpos. Uma abordagem ainda inédita neste contexto é o desenvolvimento de uma metodologia experimental eficiente para a obtenção de bibliotecas de Fab na infecção pelo vírus ZIKA (ZIKV). A infecção pelo vírus ZIKA (ZIKV) vem sendo associada à casos de microcefalia em neonatos de gestantes infectadas e síndrome de Guillain-Barré, a neutralização viral baseada em anticorpos dirigidos contra epítopos específicos é pouco conhecida, a maioria dos estudos se referem a outros Flavivirus. A resposta imunológi... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Conformational libraries of antibodies have become important tools for the scientific investigation of immunoglobulins expressed under different biological situations, including pathological conditions. The knowledge and structural elucidation of differential antibodies produced during distinct pathological conditions may be fundamental for intervention in the pathophysiology of diseases, including differential diagnosis, biomarker identification, immunoglobulin-based therapy for pathogen blocking, and constitution of antibody panels for identifying epitopes for vaccine advancement. Currently, the greatest difficulty reported in the scientific literature is the lack of methodological information for the experimental phases necessary for in vitro construction of Fab (Antigen Binding Fragment), an essential substrate for antibody library construction. In this context, an efficient experimental methodology for obtaining Fab libraries in ZIKA (ZIKV) virus infection is as yet unreported. ZIKV infection has been associated with cases of microcephaly in neonates of infected pregnant women and those with Guillain-Barré syndrome. However, viral neutralization based on antibodies directed against specific epitopes is not well understood, therefore, most studies have been carried out on other Flaviviruses. The humoral immune response is essential for protection against Flaviviruses; hence, it is important to understand the mechanisms of the host immune system against this virus. Thus, t... (Complete abstract click electronic access below) / Mestre

Early detection of illness is essential in preventing symptoms from escalating and infectious diseases from spreading. Electrochemical biosensors are a promis- ing tool in healthcare detection. Previously, the collaboration between the Arold and Inal labs has led to the design of organic electrochemical transistors (OECT) capable of rapidly detecting coronavirus in saliva by using nanobody constructs as biorecognition units. In this project, I aimed to prove the versatility of nanobody- functionalized OECT biosensors in detecting other relevant viruses, specifically, Zika and Dengue. Both viruses pose a risk to multiple populations around the world, including the Kingdom of Saudi Arabia. I designed and produced nanobod- ies that are reported to bind to the NS1 glycoprotein, which is released by Zika and Dengue into the blood of the patient. Then, I confirmed the binding of the nanobodies to their associated targets. I also developed a robotic liquid handling script to automate the biosensing operations. Ultimately, this project aims to support the design of a multiplex OECT biosensor for blood-borne pathogens.

Prenatal exposure to Zika virus (ZIKV) can result in microencephaly and congenital Zika syndrome but why some brain cells and structures are initially spared by the virus is unknown. Here, a novel murine model of ZIKV infection incorporating in utero electroporation with cell type specific promotors was used to identify the time course of ZIKV infection and to determine which neural precursor cells are initially infected or spared. In vivo time course studies revealed early presence of ZIKV in apical radial glial cells (aRGCs) while infection of basal intermediate progenitor cells climbed after three days of virus exposure. ZIKV-exposed fetal brains exhibited microencephaly as early as 1 day post injection, caused by apoptosis and reduced proliferation, and this change in brain size persisted until birth regardless of developmental age at infection. During infection, 60% of aRGC basal fibers were perturbed while 40% retained normal morphology, indicating that aRGCs are not uniformly vulnerable to ZIKV infection. To evaluate this heterogeneous vulnerability, we generated cell type-specific fate mapping plasmid probes using a previously published single cell RNA-Seq dataset on the E15.5 mouse neocortical wall. The results indicate that one class of aRGC preferentially expresses the putative ZIKV entry receptor AXL, and that these cells are more vulnerable to ZIKV infection than the other aRGC subtypes with low AXL expression. Together, these data highlight important temporal and cellular details of ZIKV fetal brain infection and may be important for prevention strategies and for management of congenital Zika syndrome.

People are afraid of contagious diseases. The thought that disease can spread throughout an entire population tends to make people wary of their interactions with their surrounding environment. Hearing about, or even seeing pictures of mosquitoes can make people squeamish or even stimulate an itch. Throughout the ages, the reaction to contagious diseases has been to quarantine and isolate. From the bubonic plague to the 1918 “Spanish” flu, the protocol was to quarantine those infected and isolate the rest. It may be this practice that inspired such precautions be taken by the public. Often these precautions are not warrantless and come to be second nature: Don’t get too close to sick people who appear to sneeze or cough often; cover your mouth when sneezing or coughing; wash your hands frequently. In recent years, the world has encountered new outbreaks from not so new diseases: 2002 SARS. 2009 “Swine” Flu. 2014 both Measles and Ebola. 2016 Zika. To the public, it seems that the next disease may strike at any moment. It is often the government’s duty to intervene and alleviate the damages. This thesis examines the legal aspects of the Zika virus and how past regulations have affected the spread of contagious diseases. In addition, it will examine past outbreaks of different diseases: how the country reacted, what policies were enacted, and how they relate to the current case of the Zika Virus.

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The world has proven unprepared for deadly viral outbreaks. Designing antiviral drugs and strategies requires a firm understanding of the interactions taken place between the proteins of the virus and human proteins. The current computational models for predicting these interactions consider only single viruses for which extensive prior knowledge is available. The two prediction frameworks in this dissertation, DeNovo and DeNovo-Human, make it possible for the first time to predict the interactions between any viral protein and human proteins. They further helped to answer critical questions about the Zika virus. DeNovo utilizes concepts from virology, bioinformatics, and machine learning to make predictions for novel viruses possible. It pools protein-protein interactions (PPIs) from different viruses sharing the same host. It further introduces taxonomic partitioning to make the reported performance reflect the situation of predicting for a novel virus. DeNovo avoids the expected low accuracy of such a prediction by introducing a negative sampling scheme that is based on sequence similarity. DeNovo achieved accuracy up to 81% and 86% when predicting for a new viral species and a new viral family, respectively. This result is comparable to the best achieved previously in single virus-host and intra-species PPI prediction cases. DeNovo predicts PPIs of a novel virus without requiring known PPIs for it, but with a limitation on the number of human proteins it can make predictions against. The second framework, DeNovo-Human, relaxes this limitation by forcing in-network prediction and random sampling while keeping the pooling technique of DeNovo. The accuracy and AUC are both promising ($>85%$, and $>91%$ respectively). DeNovo-Human facilitates predicting the virus-human PPI network. To demonstrate how the two frameworks can enrich our knowledge about virus behavior, I use them to answer interesting questions about the Zika virus. The research questions examine how the Zika virus enters human cells, fights the innate immune system, and causes microcephaly. The answers obtained are well supported by recently published Zika virus studies. / Ph. D. / When a virus attacks a human body, it disturbs the host cells by interacting with their proteins. Identifying these interactions is key to fighting the virus. In this dissertation, I developed two computational tools to identify the interactions for any virus infecting the human. I further used these tools to answer interesting questions about the Zika virus behavior. The results are in agreement with recently published experimental studies about the virus.

Depuis sa réémergence durant l'épidémie de 2013-2017 dans les régions du Pacifique et des Amériques, le virus Zika a été associé à plusieurs complications neurologiques chez l'adulte. L'immunité innée représente une composante importante de la réponse immunitaire au sein du système nerveux central (SNC). Nous avons donc postulé que les microglies, macrophages résidents du SNC, jouent un rôle actif dans le développement de la réponse initiale à l'infection du cerveau mature par le virus Zika. Nous avons utilisé un modèle murin non létal, déficient pour des composantes de l'immunité innée, pour étudier le rôle des microglies dans la pathogénèse et la réponse immunitaire lors de l'infection du SNC. Les souris infectées présentaient une virémie et une charge virale cérébrale élevées sans manifestations cliniques de l'infection. Une analyse immunohistochimique a montré que le virus Zika était distribué dans plusieurs régions du cerveau, particulièrement dans l'hippocampe dorsal. Dans cette région, le nombre de microglies est demeuré stable après l'infection, mais elles montraient des caractéristiques morphologiques compatibles avec un état de réactivité. Une analyse ultrastructurelle en microscopie électronique a révélé que les microglies présentaient une activité phagocytaire et une digestion extracellulaire accrues durant l'infection. La déplétion pharmacologique des microglies a provoqué une augmentation de la réplication virale et les astrocytes présentaient une activité phagocytaire compensatoire. Dans l'ensemble, ces résultats montrent que les microglies sont impliquées dans le contrôle de la réplication du virus Zika et dans son élimination phagocytaire au sein du SNC de souris jeunes adultes immunodéficientes. / Since its re-emergence during the 2013-2017 epidemic across the Pacific and the Americas, Zika virus has been associated with several neurological complications in adults. Because innate immunity is an important component of the immune response within the central nervous system (CNS), we postulated that microglia, the resident macrophages of the CNS, play an active role in the development of the initial response to infection of the mature brain with Zika virus. We used a non-lethal mouse model deficient in components of innate immunity to study the role of microglia in the pathogenesis and immune response of Zika virus infection of the CNS. Infected mice developed elevated viremia and brain viral load without showing clinical signs of infection. Immunohistochemical analysis showed that Zika virus was distributed in several regions of the brain, predominantly in the dorsal hippocampus. In this region, the number of microglia remained steady after infection with Zika virus, but microglia adopted morphological characteristics consistent with a state of reactivity. Ultrastructural analysis by electron microscopy revealed that microglia exhibited increased phagocytic activity and extracellular digestion during infection. Pharmacological depletion of microglia caused an increase in viral replication and astrocytes exhibited compensatory phagocytic activity. Taken together, these results show that microglia are involved in the control of Zika virus replication and its phagocytic elimination within the brain of immunodeficient young adult mice.

O Zika vírus (ZIKV) é um vírus RNA de fita única, pertencente à família Flaviviridae. É transmitido entre os humanos geralmente pelos mosquitos da espécie Aedes, mas transmissão via sexual, perinatal e por transfusão sanguínea também foram relatadas. Os sintomas aparecem em 20% dos indivíduos infectados e incluem febre, dor de cabeça, rash cutânea, conjuntivite, mialgia e artralgia. Em 2016, durante a grande epidemia do ZIKV pelas Américas, o interesse pelo seu diagnóstico rápido se intensificou, devido a relação do vírus com o aumento da incidência de casos da síndrome de Guillain-Barré em adultos e da microcefalia em recém nascidos de mulheres grávidas infectadas. De acordo com o CDC (Center for Disease Control and Prevention) o diagnóstico dos pacientes sintomáticos deve ser realizado através da detecção dos ácidos nucleicos do vírus por PCR (Polymerase chain reaction) em amostras pareadas de sangue e urina. Estudos recentes têm postulado que a saliva é uma alternativa importante para detecção do ZIKV. A saliva requer menor complexidade no processamento quando comparada ao sangue, simplificando a reação. A amplificação Isotérmica mediada por Loop (LAMP) é um teste sorológico de alta sensibilidade e especificidade para detectar rapidamente DNA ou RNA de patógenos, incluindo o ZIKV. O fato de não requerer ciclos térmicos como o PCR, faz do LAMP uma reação mais simples, rápida e mais econômica por exigir menos energia. O objetivo deste estudo foi de avaliar e comparar a eficácia da saliva e da urina em diagnosticar a infecção pelo Zika vírus em indivíduos na fase aguda da doença, através da detecção do RNA viral por meio do LAMP. Ao todo, 131 amostras (68 saliva e 63 urina) de 69 indivíduos brasileiros apresentando sinais e sintomas específicos e confirmados positivamente para o ZIKV através da análise do sangue por PCR, foram coletadas e analisadas por LAMP. A média de idade dos indivíduos foi de 34,7 (±13,6), sendo 46 (66,7%) do sexo feminino. Das 68 amostras de saliva analisadas por LAMP, 45 (66,2%) foram positivas para o ZIKV com o Tempo de positividade (Tp) médio de 13,5 minutos. Enquanto que das 63 amostras de urina, 25 (39,7%) foram positivas com o Tp médio de 15,8 minutos. A saliva pôde diagnosticar mais indivíduos (p=0.0042) e em menor Tp (p=0.0176) quando comparada à urina. A saliva demonstrou ser uma alternativa viável no diagnóstico da infecção do ZIKV, em indivíduos na fase aguda da doença, através do LAMP. Nossos achados contribuem para o conhecimento do comportamento do Zika vírus no organismo, uma vez que pouco se conhece em relação à excreção do ZIKV na saliva. / Zika virus (ZIKV) is a single-stranded RNA virus, member of the Flaviviridae family. It is transmitted among humans usually by Aedes mosquito species, but sexual transmission, perinatal and blood transfusion have also been reported. Symptoms appear in 20% of infected individuals and include fever, cutaneous rash, headache, conjunctivitis, myalgia and arthralgia. In 2016, during the Americas ZIKV outbreak, the interest in a rapid diagnosis intensified due to a sudden increase in cases of Guillain-Barré syndrome in adults and microcephaly in newborns of infected pregnant women related with ZIKV. According to CDC (Center for Disease Control and Prevention) the diagnosis of symptomatic patients should be done through nucleic acid detection by PCR (Polimerase Chain Reaction) in paired samples of blood and urine. Recent studies have reported that saliva can be an important alternative to detect ZIKV. Saliva requires less processing than blood, which greatly simplifies the assay process. Loop-mediated Isothermal Amplification (LAMP) is a molecular test with high sensibility and specificity for rapid detection of DNA or RNA of pathogens, including ZIKV. The fact that LAMP does not require thermal cycling makes the assay simple, fast and cost effective compared to PCR assay. The aim of this study was to evaluate the efficacy of saliva and urine to diagnose ZIKV infection in subjects during the acute phase, through ZIKV RNA detection by LAMP. A total of 131 samples (68 saliva and 63 urine) from 69 subjects in the acute phase of ZIKV infection and confirmed positive for ZIKV by blood analysis through PCR were collected and analyzed by LAMP. The mean age of the individuals was 34.7 (±13,6) years old, of whom 46 (66.7%) were females. From the 68 saliva samples, 45 (66.2%) were positive for ZIKV with an average time to positivity (Tp) of 13.5 minutes, and from the 63 urine samples, 25 (39.7%) were positive with an average Tp of 15.8 minutes. Saliva detected more samples (p=0.0042) and had faster Tp (p=0.0176) as compared to urine. Thus, saliva proved to be a feasible alternative for diagnosis of ZIKV infection during the acute phase by LAMP. The findings of this study can contribute to the knowledge of the Zika virus behavior in the human organism, since this issue is not totally understood.

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.