Les IDPs sont des protéines dépourvues de structure 3D unique en solution et en l'absence de leur(s) partenaire(s). Ces protéines possèdent des propriétés d'interactions avec leurs partenaires uniques.L'extrêmité C-terminale de la nucléoprotéine du virus de la rougeole, NTAIL, est une IDP. NTAIL interagit avec XD, le domaine C-terminal globulaire de la phosphoprotéine virale, via la box2 qui est un a-MoRE. Cette interaction permet le recrutement de la protéine L afin de former la polymérase virale.J'ai pu montrer que la contribution des différents résidus au sein de l'a-MoRE dépend de l'orientation de leur chaîne latérale, et que la substitution d'un seul acide aminé crucial a des effets dramatiques sur la réplication virale.Les IDPs conservent un désordre résiduel non négligeable au sein du complexe. Cela se manifeste par la présence des régions « fuzzy » de part et d'autres du MoRE. Nous avons montré que la région « fuzzy » en amont de la box2 inhibe l'établissement de l'interaction entre NTAIL et ses partenaires.Lors de l'interaction avec leurs partenaires les IDPs subissent en général un gain de structure. Le repliement associé à l'interaction peut avoir lieu avant ou après interaction. Des études précédentes ont montré l'existence d'une pré-structuration partielle de l'alpha-MoRE de NTAIL. L'interaction entre NTAIL et XD a été étudiée et a permis de conclure que NTAIL se replie selon un mécanisme de repliement induit.Dans leur ensemble, ces études contribuent à éclaircir les mécanismes moléculaires qui gouvernent la reconnaissance de partenaires par les IDPs. / IDPs are proteins devoided of a unique and stable 3D structure in solution and in the absence of their partners. Those proteins possess unique properties, as well as mechanisms of interaction with their partners.The C-terminal region of the nucleoprotein of measles virus, NTAIL, is an IDP and interacts with XD, the globular C-terminal domain of the viral phosphoprotein, via the box2 region that is an (alpha-MoRE). This interaction allows to recruit the L protein in order to form the viral polymerase.The aim of my work was to characterize the molecular basis of NTAIL-XD interaction. I was able to show that the contribution of the amino acids among box2 depends on the orientation of their lateral chain, and that the substitution of one single amino acid has drastic effect upon the viral replication.IDPs keep a non-negligible amount of residual disorder among the complex. This fuzziness can have multiple forms, like the presence of fuzzy regions from either side of the MoRE. The impact fuzzy regions have within the complex is not well known. We demonstrated that the fuzzy region upstream box2 inhibits the settlement of the interaction between NTAIL and its partners.When interacting with their partners, IDPs generally undergo a folding associated with binding that can take place either before or after the interaction. The interaction between NTAIL and XD was investigated and monitored by fluorescence kinetic measurements, using variants bearing a tryptophan substitution. We concluded without any doubt that NTAIL folds under an induced folding mechanism.Those studies together contribute to enlighten the molecular mechanisms that govern partner recognition by the IDPs.

L’ordre viral des Mononegavirales contient de nombreux virus pathogènes tels que le virus de la rougeole, le virus de la rage, le virus des oreillons ou encore le virus Ebola. Ces virus mettent tous en place des mécanismes moléculaires similaires, notamment concernant la synthèse des ARN viraux. Le complexe polymérase, composé de la polymérase virale (L) et de la phosphoprotéine (P), possède un fonctionnement encore obscur et unique dans le monde du vivant, notamment car il utilise une matrice enchâssée dans une gaine protéique. La formation de ce complexe a été étudiée et la protéine chaperon HSP90 (« Heat Shock Protein of 90 kD ») s’est révélée nécessaire à la formation du complexe. L’inhibition de l’activité d’HSP90 entraine l’ubiquitination et la dégradation de la protéine L par le protéasome. Les protéines P et HSP90 sont toutes les deux nécessaires au repliement de la protéine L et à la formation d’un complexe P-L stable, soluble et fonctionnel. Les domaines de P impliqués dans la formation du complexe, ont également été cartographiés et révèlent des interactions complexes entre P et L, mêlant liaison, stabilisation, repliement et fonction. Enfin, une interaction entre P et la protéine virale C, connue pour inhiber la synthèse des ARN viraux, a été identifiée, cartographiée et ouvre des perspectives quant aux mécanismes moléculaires sous-jacents à son effet inhibiteur. / The Mononegavirales order contains several pathogens like measles, rabies, mumps and Ebola viruses. These viruses share numerous homologous molecular mechanisms and in particular they have a highly conserved RNA synthesis machinery that is unique in the living world. Indeed, the polymerase complex, composed of the polymerase (L) and the phosphoprotein (P), uses a template of RNA recovered by a sheath made of nucleoproteins. The formation of the complex was investigated and the chaperone protein HSP90 (Heat Shock Protein of 90 kD) was shown to be required for the formation of the complex. The inhibition of HSP90 activity induces the ubiquitinylation and the degradation of the L protein by the proteasome. Both P and HSP90 are required to form stable, soluble and functional polymerase complexes. The domains of P involved in the formation of the complex have been mapped and they show that the interplay between P and L is complex with at least three identified functions: binding, folding and function of the polymerase complex. Finally, an interaction between P and the viral C protein, known to inhibit the viral RNA synthesis, have been identified, mapped and allows new perspectives concerning the molecular mechanism underlying its inhibitory effect.

Diversos trabalhos têm demonstrado a presença de peptídeos bioativos em hemolinfa de insetos e seu potencial uso como agentes terapêuticos. Este trabalho buscou identificar e isolar proteínas da hemolinfa de Lonomia obliqua com ação antiviral. A adição de hemolinfa antes da infecção reduziu o título de poliovírus de 1,5x107 TCID50/mL no cultivo controle para 4x105 TCID50/mL e em aproximadamente 100 vezes o título do sarampo. Após cromatografia de gel filtração, foram obtidos 3 pools de proteína. O pool responsável pela ação antiviral foi identificado (Pool 2), uma vez que reduziu em 87 vezes o título de poliovírus, e de 1,6x106 TCID50/mL para 2,7x105 TCID50/mL o título do sarampo. O Pool 2 foi fracionado por cromatografia de troca iônica. A fração responsável pela ação antiviral da hemolinfa de Lonomia obliqua frente ao poliovírus e sarampo foi identificada (RQ 3-4), esta proteína, de aproximadamente 20 kDa, tem ação sobre vírus envelopado (sarampo) e vírus desprovido de envelope viral (poliovírus). / Several works have demonstrated the presence of bioactive peptides in insect hemolymph and their potential use as therapeutic agents. This work sought to identify and purify proteins from Lonomia obliqua hemolymph with antiviral activity. The analyses demonstrated that the best time for hemolymph addition in the cell culture is before the infection, the poliovirus titer had reduced from 1.5x107 TCID50/mL to 4x105 TCID50/mL and for measles, the virus titer was about 100 times lower than the control. After gel filtration chromatography, the hemolymph was divided into 3 pools of protein. The pool responsible for the antiviral activity was identified (Pool 2), once it reduced to 87 times the poliovirus titer, and the measles titer from 1.6x106 TCID50/mL to 2.7x105 TCID50/mL. The Pool 2 was fractionated by ion-exchange chromatography. The fraction from Lonomia obliqua hemolymph responsible for the antiviral activity was identified - RQ 3-4, this protein of approximately 20 kDa, has antiviral effect on enveloped virus (measles) and on non enveloped virus (poliovirus).

Abstract Background Measles is a serious infectious disease that contributes significantly to the burden of disease in many developing countries. In most developed nations, such as Canada, endemic transmission of measles has been declared eliminated thanks to rigorous vaccination programs, but isolated outbreaks of the disease continue to happen. Therefore, a thorough understanding of the factors contributing to these outbreaks is necessary. Objectives There were two main objectives of this thesis. The first objective was to assess the geospatial distribution of reported measles cases in Ontario with a goal of identifying clusters of reported measles. For this objective, the main hypothesis was that measles cases would not be randomly distributed across Ontario and instead would cluster in certain regions. The second objective was to explore some of the factors that may be associated with measles clusters. For this objective, the main hypothesis was that the proportion of immigrants, population density, low-income prevalence and education level would be associated with measles clusters. Methods The first objective was achieved through a thorough geospatial analysis using SaTScan and R. Individual forward sortation areas were used as the spatial unit of analysis. The analysis leveraged data from multiple sources: 2016 Census data, Ontario measles cases data from iPHIS from 2008 to 2019, a shapefile of all forward sortation areas in Canada from Statistics Canada and centroid coordinates of forward sortation areas that were obtained using web scrapping techniques on the geolocation service of Natural Resources Canada. The maximal window size of the geospatial analysis was chosen using the maximum clustering heterogeneous set-proportion technique. The geospatial analysis was run with 99,999 Monte Carlo repetitions under a Poisson distribution using the purely spatial analysis. The Ontario population from the 2016 Census was used as the population at risk. Any cluster with a p ≤ 0.05 was deemed statistically significant. The second objective was achieved through a case-control study: Forward sortation areas that were within statistically significant measles clusters were considered as cases and the rest of the forward sortation areas were considered as controls. Demographic data necessary to assess the factors of interest were extracted from the 2016 Census. A univariable logistic regression model was run to compute the odds ratio and test the association between the factors of interest and measles clusters. 95% confidence intervals were computed for each odds ratio. Data-curation techniques and data analysis were performed in R 4.0.4. Results From 2008 through 2019, 178 measles cases were identified. 82% of cases lacked necessary vaccination or vaccination records against measles, 35% of cases were linked to traveling outside of Ontario, 20% of cases reported being in contact with a known case, and 72% of cases were less than 5 years old or older than 21. Ten measles clusters were identified of which six were deemed statistically significant. These six significant clusters represented 7% of the population at risk but contained nearly 40% of all reported measles cases between 2008 and 2019. Measles clusters had a strong association with the proportion of immigrants living within them, population density and prevalence of low-income. No association was found between education level and measles clusters. Conclusion The results indicate that most measles cases in Ontario are unvaccinated or lack proof of vaccination; arise through secondary transmission within the province; arise from undetected transmission; and are adults or infants. Additionally, it is possible to see that the risk of reported measles cases is not randomly distributed across the province, but instead measles cases tend to cluster in certain regions. Such clusters tend to be characterized by specific population-level factors that may be contributing to the risk of reported measles. Targeted and equitable interventions are needed as we continue on the path to eradication.

This master thesis is focused on decreasing levels of vaccination as a new health risk. In recent years it has become a new trend that we can observe in European countries. Low vaccination levels amongst population are one of the reasons for new epidemics or pandemics happening. When immunization rises above 95 % then a collective immunization comes into effect. Collective immunization lowers the chances of diseases spreading. For this reason, World health organization supports immunization and tries to make immunization affordable reachable for everyone everywhere. This these analyses measles which can be stopped by vaccinating the population. Therefore, the World health organization prepares strategic plans aimed at eradication of this disease. Each member state has a task to apply these plans in their specific environment. This thesis focuses on the Czech Republic and its implementation of strategic plans happening 2005-2010 and 2011-2020. This thesis is structured into 5 chapters focused on theory of international relations, hesitancy to vaccinate, strategic plans of World health organization and the Czech Republic.

Measles is a potentially deadly illness that has been declared no longer endemic in the United States of America since 2000.1 However, in the past few years, imported cases of the measles have continued to cause hospitalization and deaths among those citizens who remain unvaccinated, or have waning immunity, against measles, especially children. The Measles, Mumps and Rubella (MMR) vaccine has been available since 1963 and is routinely given to children in the first two years of life.1 Endemic cases of measles are increasing each year, specifically in undervaccinated communities. In 2018-2019, there was an outbreak of measles in the Williamsburg neighborhood of Brooklyn, New York. Investigating the knowledge, attitudes and opinions on the measles virus and the MMR vaccine in the Williamsburg neighborhood may facilitate discussions that could increase the vaccination rate among its population, as well as elucidate more effective strategies for vaccination in the future. Comparing attitudes from the Williamsburg neighborhood with a population across the Hudson River, in the East Village, which has previously had higher rates of vaccination, could shed light on how to target and tailor vaccination campaigns to different populations in New York City moving forward.2–4

Analysis of Spontaneous Adverse Events Reports of Measles, Mumps, and Rubella Vaccine Author: Jana Kulhavá Supervisor: PharmDr. Eva Zimčíková, Ph.D. Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Social and Clinical Pharmacy Keywords: vaccination, measles, mumps, rubella, adverse events reports Introduction: The MMR vaccine is a combined vaccine used to vaccinate children against measles, mumps and rubella. Spontaneous reporting of adverse reactions is an important source of information to identify potential risks of medicinal products. Objective: The aim of this diploma thesis is the analytical evaluation of spontaneous reports of suspected adverse reactions after vaccination with MMR vaccine registered in the database of the State Institute for Drug Control during the period 2004 to 2017. Methods: The data were analyzed in Microsoft Excel spreadsheet software using descriptive statistics methods. The reported adverse reactions were classified into appropriate organ system classes according to the MedDRA Glossary of Medical Terminology. The expectability and severity of adverse reactions were assessed. Results: A total of 805 cases of suspected adverse reactions were reported between 2004 and 2007, which included 2,812 adverse reactions. Most suspected adverse...

Die subakute sklerosierende Panenzephalitis (SSPE), eine durch das Masernvirus (MV) verursachte sogenannte „ slow virus “ Infektion des zentralen Nervensystems, ist eine progrediente chronische Erkrankung, die zum Tod führt und bisher medikamentös nicht heilbar ist. Da die RNAi-Strategien grundsätzlich zur Inhibition von Viren in Säugetierzellen geeignet sind, stellt die RNAi eine Möglichkeit dar, die Infektion auf molekularer Ebene anzugreifen. Dafür wurden verschiedene miRNA-Expressionskassetten, welche gegen zwei Sequenzen im MV- Hämagglutinin-Gen (H) und sechs Sequenzen im MV-Nukleokapsid-Gen (N) gerichtet sind, konstruiert und in MV infizierte Zellen eingebracht. Diese miRNAExpressionskassetten wurden auf zwei verschiedenen Wegen in die Zelle eingebracht: Zum einen wurden sie über ein miRNA-Expressionsplasmid (pmiR), welches in die Zellen transfiziert wird, transient exprimiert; zum anderen wurden sie durch virale Vektoren (HIV, SIV und MoMLV) stabil in die Zellen transduziert. Dies ermöglicht die Integration der miRNAExpressionskassette in das Genom der Zelle und dadurch die Expression der miRNAs für einige Wochen. In erster Linie zielt die Wirkung der RNA-Interferenz auf die Degradierung der spezifischen MV-mRNAs. Diese Degradierung konnte mit Hilfe quantitative Reverse Transkription real time-PCR (qRT-PCR) nachgewiesen werden. Die transiente Expression der verschiedenen miRNAs gegen das MV-N-Gen bzw. MV-H-Gen führte in jedem Fall zu einer Reduktion der viralen genspezifischen mRNAs. Die Reduktion der MV spezifischen mRNA betrug 99,8% für das MV-N-Gen und 91,2% für das MV-H-Gen. Die Wirkung der RNA-Interferenz zielt am Ende auf die Reduktion der neu gebildeten infektiösen Viruspartikel und ihrer Verbreitung in der Zellkultur, welche die spezifische miRNA gegen MV-N oder MV-H exprimiert. Dieser Effekt konnte nur durch Plaque-Assay überprüft werden. Die Plaque-Assays, die mit den Überständen der miRNA-behandelten Zelllinien durchgeführt wurden, zeigten ebenfalls eine Reduktion der neu gebildeten infektiösen MV-Partikeln von 97,6% für die miRNA gegen das MV-H-Gen und 99,0 % für die miRNA gegen das MV-N-Gen. Die intrazelluläre Expression der miRNAs führte zu einer Hemmung der Virus-Ausbreitung in MV-infizierten Zellen. Die Reduktion betrug hier durch die Expression der miRNA-N10 98,8% und durch die Expression der miRNA-H2 80,0%. Hier zeigte sich, dass die Inhibition der viralen Proteinsynthese durch den RNAi-Mechanismus auch die Verbreitung der MVInfektion durch Zell-Zell-Fusion behindern kann. Dies zeigte sich durch die verringerte Bildung von Plaques bzw. Synzytien in miRNA-behandelten Zelllinien. Die vorliegende Arbeit zeigte, dass RNAi effektive gegen MV-Infektion in Zellkultur eingesetzt werden kann. Als nächster Schritt sollte daher dieser RNAi-Effekt im etablierten Tiermodell ausgetestet werden.

Measles virus (MV) can cause severe acute diseases as well as long-lasting clinical deteriorations due to viral-induced immunosuppression and neuronal manifestation. How the virus enters the brain and manages to persist in neuronal tissue is not fully understood. Various mutations in the viral genes were found in MV strains isolated from patient brains. In this study, reverse genetics was used to introduce mutations in the fusion, matrix and polymerase genes of MV. The generated virus clones were characterized in cell culture and used to infect rat brain slice cultures. A mutation in the carboxy-terminal domain of the matrix protein (R293Q) promoted the production of progeny virions. This effect was observed in Vero cells irrespective of the expression of the signaling lymphocyte activation molecule (SLAM). Furthermore, a mutation in the fusion protein (I225M) induced syncytia formation on Vero cells in the absence of SLAM and promoted viral spread throughout the rat brain slices. In this study, a solid ex vivo model was established to elucidate the MV mutations contributing to neural manifestation.

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