Investigation des interactions chez les Flavivirus dans le but de découvrir des nouvelles cibles thérapeutiques

Abstract : Mosquito-borne Flaviviruses include important human pathogens such as dengue virus, Zika virus, Yellow Fever virus, and West Nile virus. Although most infections are asymptomatic and most symptomatic cases are associated with a flu-like illness, a small fraction of Flavivirus infections can lead to severe illness, including encephalitis and hemorrhagic fever which may be fatal. Despite several decades of research efforts, no specific antiviral drugs are currently available to treat Flavivirus infections. Therefore, new potential targets for the discovery and/or design of antiviral therapies are needed. Identifying and understanding virus-host interactions has been proposed to be a promising avenue for the development of novel strategies to treat or prevent infections. Here, Flavivirus-host interactions were investigated using RNA sequencing as well as SILAC/MS, and the coding transcriptomes and proteomes of cells infected with three different Flaviviruses, namely Kunjin virus, Zika virus and Yellow Fever virus, was compared to those of mock-infected cells. Approximately 300-500 genes and 550-700 proteins were found to be differentially expressed during Flavivirus infections, and roughly 600-900 modulations in alternative splicing of host transcripts were identified. Moreover, 51 genes, 39 alternative splicing events and 57 proteins were found to be affected in all three Flavivirus infections. Several of these genes/proteins would be interesting candidates to study further for their roles in viral infections and their potential to be targeted to suppress viral replication. Another attractive target for the development of therapeutic compounds are protein-protein interactions. Here, the West Nile virus NS3:NS5 interaction, which is essential for viral replication, has been characterized using site-directed mutagenesis based on an interaction model that was developed in silico. Seven residues on the surface of the NS3 protein, clustered in two ‘hotspots’, were found to be critical for viral replication, possibly by mediating the NS3:NS5 interaction. These two ‘hotspots’ could be interesting targets for future anti-flaviviral drug development. In conclusion, the work presented in this thesis has highlighted several human genes/proteins as well as two regions on the surface of the viral protein NS3 that deserve further study to determine their potential to be targeted for the development of antiviral therapies.Les flavivirus comprennent d'importants agents pathogènes humains tels que le virus Zika, le virus de la fièvre jaune et le virus du Nil occidental. Bien que la majorité des infections soient asymptomatiques et la plupart des cas symptomatiques soient associés à une maladie pseudo-grippale, un faible pourcentage des infections par les flavivirus peut entraîner des maladies graves qui peuvent être mortelles. Malgré plusieurs décennies d'efforts de recherche, aucun médicament antiviral n'est actuellement disponible pour traiter ces infections. Par conséquent, de nouvelles cibles potentielles pour la découverte et/ou la conception de thérapies antivirales sont nécessaires. L'identification et la compréhension des interactions virus-hôte ont été proposées comme une voie prometteuse pour le développement de nouvelles stratégies pour traiter ou prévenir les infections. Ici, les interactions entre trois flavivirus (Kunjin, Zika, fièvre jaune) et leurs cellules hôtes ont été étudiées à l'aide du séquençage de l'ARN ainsi que de la protéomique quantitative (SILAC). Plusieurs centaines de gènes et protéines se sont avérés être exprimés de manière différentielle lors de ces infections, et environ 600 à 900 modulations dans l'épissage alternatif des transcrits cellulaires ont été identifiées. Plusieurs de ces gènes/protéines seraient des candidats intéressants à étudier davantage pour leurs rôles dans les infections virales et leur potentiel à être ciblés pour supprimer la réplication virale. Une autre approche intéressante pour le développement de composés thérapeutiques est de cibler les interactions protéine-protéine. Ici, l'interaction entre les protéines NS3 et NS5 du virus du Nil occidental, qui est essentielle pour la réplication virale, a été caractérisée. Sept résidus à la surface de la protéine NS3, regroupés en deux petites régions, se sont révélés critiques pour la réplication virale, possiblement en médiant l'interaction NS3:NS5. Ces deux petites régions pourraient être des cibles intéressantes pour le développement futur de médicaments contre les flavivirus. En conclusion, les travaux présentés dans cette thèse ont mis en évidence plusieurs gènes/protéines humains ainsi que deux régions à la surface de la protéine virale NS3 qui mériteraient d’être étudiés davantage afin de déterminer leur potentiel à être ciblés pour le développement de thérapies antivirales

El Camino de Santiago: estudio comparativo de las ofertas española y alemana

El TFG trata de la comparación de la oferta española para el Camino de Santiago con la oferta alemana. La autora ha presentado la historia del camino y varias rutas como también unas estadísticas. Después ha comparado unas ofertas en los mercados turísticos de ambos países que existen en el Camino de Santiago. En el mercado turístico alemán la autora se refiere también al cómico alemán Hape Kerkeling que publicó un libro sobre su viaje en el Camino y ha analizado la influencia en la demanda alemana. Al final da opciones para el desarrollo en el futuro.Grado en Turism

Caractérisation détaillée de l’interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive

Les Flavivirus transmis par les moustiques comme le virus du Nil occidental, le virus de la dengue, le virus de la fièvre jaune, le virus de l’encéphalite japonaise et le virus Zika constituent des préoccupations croissantes de santé publique. Ils se sont répandus dans le monde au cours des dernières décennies, et les épidémies sont devenues plus fréquentes et plus sévères. Chaque année, des millions de personnes sont infectées et environ 50 000 patients décèdent d’infections à Flavivirus. Malgré les nombreux efforts de recherche, il n’y a actuellement aucun médicament antiviral spécifique disponible, et des nouvelles stratégies antivirales sont indispensables. Comprendre comment les Flavivirus fonctionnent au niveau moléculaire aidera à découvrir des nouvelles cibles pour l'intervention thérapeutique. Les Flavivirus ont un génome d'ARN simple brin de polarité positive qui code pour trois protéines structurales et huit protéines non structurales. Seules deux des huit protéines non structurales ont des activités enzymatiques. NS3 possède un domaine protéase et un domaine hélicase, et NS5 a un domaine méthyl- et guanylyltransférase et un domaine ARN polymérase ARN-dépendante. Ensemble, ils répliquent le génome viral. Ici, nous caractérisons l'interaction entre NS3 et NS5 dans le complexe de réplication du virus du Nil occidental pendant la synthèse d’ARN de polarité positive. Un modèle d'interaction comprenant NS3, NS5 et l’ARN viral a été développé basé sur des structures cristallines connues ainsi que des activités enzymatiques des deux protéines individuelles, et ce modèle a été soumis à des simulations de dynamique moléculaire. Les interactions potentielles entre les protéines NS3 et NS5 ont été identifiées. Les résidus impliqués dans ces interactions ont été mutés dans un réplicon du virus du Nil occidental et les effets de ces mutations sur la réplication virale ont été évalués. Une région particulière à la surface de la protéine NS3 a été identifiée comme étant cruciale pour la réplication virale, très probablement parce qu'elle interagit avec NS5. Cette région pourrait être une cible attrayante pour la recherche de composés qui pourraient interférer avec l'interaction entre NS3 et NS5 et donc posséder un potentiel antiviral intéressant.Abstract : Mosquito-borne Flaviviruses like West Nile virus, Dengue virus, Yellow Fever virus, Japanese encephalitis virus, and Zika virus are increasing public health concerns. They have spread globally during the past decades, and outbreaks have recently become more frequent and more severe. Every year, millions of people are infected, and approximately 50,000 patients die from Flavivirus infections. Despite extensive research efforts, there are currently no specific antiviral drugs available, and new antiviral strategies are greatly needed. Understanding how Flaviviruses work on a molecular level will help in uncovering new points for therapeutic intervention. Flaviviruses have a single-stranded RNA genome of positive polarity that encodes three structural and eight non-structural proteins. Only two of the eight non-structural proteins have enzymatic activities. NS3 has an N-terminal protease domain and a C-terminal helicase domain, and NS5 has an N-terminal capping enzyme domain and a C-terminal RNA-dependent RNA polymerase domain. Together, they replicate the viral genome. Here we characterize the NS3:NS5 interaction within the West Nile virus RNA replicase complex during positive strand synthesis. An interaction model including NS3, NS5 and viral RNA was developed based on the known crystal structures as well as enzymatic activities of the two individual proteins, and this model was subjected to molecular dynamics simulations. Potential interactions between the NS3 and NS5 proteins were identified. Residues involved in these interactions were mutated in a West Nile virus replicon, and the effects of these mutations on viral replication were evaluated. One particular region on the surface of the NS3 protein was identified to be crucial for viral replication, most likely because it mediates the interaction with NS5. This region might be an attractive target for the search of compounds that could interfere with the NS3:NS5 interaction and therefore possess an interesting antiviral potential

What contributes to the long-term implementation of an evidence-based early childhood intervention: a qualitative study from Germany

BackgroundRigorous research trials have demonstrated that early childhood interventions can reach socially disadvantaged families and can have a lasting impact on the healthy development of their children. However, little is known about the internal and contextual factors that contribute to the long-term implementation of such interventions. In this study, we investigated the development of the home visiting program Pro Kind. The program was adapted from the evidence-based US-American Nurse-Family Partnership program and was implemented in Germany in 2006. Using an exploratory approach, we examined factors contributing to the long-term implementation of this program.MethodsQualitative interviews with program implementers (midwives, social workers, program managers) of the Pro Kind program and key stakeholders in two cities in Germany were conducted. Interview guides were developed to assess participants' perceptions and experiences on how the program had developed over time internally and in the interaction with its environment. Data were collected between March and September 2021. Drawing on the Consolidated Framework for Implementation Research (CFIR), data was coded according to the principles of thematic analysis.ResultsA total of 25 individuals (11 program implementers, 14 key stakeholders) were interviewed. The identified factors related to three out of five domains of the CFIR model in our analysis. First, regarding the intervention characteristics, the evidence of effectiveness and the relative advantage of the implementation of the program compared to similar interventions were viewed as contributors to long-term implementation. However, the program's adaptability was discussed as a constraining factor for reaching the target group. Second, concerning the inner setting, stakeholders and program implementers perceived the implementation climate, the leadership engagement and the program's size as relevant factors for networking strategies and program visibility. Third, as part of the outer setting, the degree of networking with external stakeholders was highlighted of great importance for the program.ConclusionsWe identified several factors of particular importance for the long-term implementation and sustainability of an early childhood intervention at the practice level, particularly in the local context in Germany. These findings should inform the design of impactful, scalable, and sustainable early childhood interventions targeting disadvantaged families

S100A9 is indispensable for survival of pneumococcal pneumonia in mice

S100A8/A9 has important immunomodulatory roles in antibacterial defense, but its relevance in focal pneumonia caused by Streptococcus pneumoniae (S. pneumoniae) is understudied. We show that S100A9 was significantly increased in BAL fluids of patients with bacterial but not viral pneumonia and correlated with procalcitonin and sequential organ failure assessment scores. Mice deficient in S100A9 exhibited drastically elevated Zn$^{2+}$ levels in lungs, which led to bacterial outgrowth and significantly reduced survival. In addition, reduced survival of S100A9 KO mice was characterized by excessive release of neutrophil elastase, which resulted in degradation of opsonophagocytically important collectins surfactant proteins A and D. All of these features were attenuated in S. pneumoniae-challenged chimeric WT→S100A9 KO mice. Similarly, therapy of S. pneumoniae-infected S100A9 KO mice with a mutant S100A8/A9 protein showing increased half-life significantly decreased lung bacterial loads and lung injury. Collectively, S100A9 controls central antibacterial immune mechanisms of the lung with essential relevance to survival of pneumococcal pneumonia. Moreover, S100A9 appears to be a promising biomarker to distinguish patients with bacterial from those with viral pneumonia. Trial registration: Clinical Trials register (DRKS00000620)

Assessing Implicit Odor Localization in Humans Using a Cross-Modal Spatial Cueing Paradigm

Navigation based on chemosensory information is one of the most important skills in the animal kingdom. Studies on odor localization suggest that humans have lost this ability. However, the experimental approaches used so far were limited to explicit judgements, which might ignore a residual ability for directional smelling on an implicit level without conscious appraisal.A novel cueing paradigm was developed in order to determine whether an implicit ability for directional smelling exists. Participants performed a visual two-alternative forced choice task in which the target was preceded either by a side-congruent or a side-incongruent olfactory spatial cue. An explicit odor localization task was implemented in a second experiment.No effect of cue congruency on mean reaction times could be found. However, a time by condition interaction emerged, with significantly slower responses to congruently compared to incongruently cued targets at the beginning of the experiment. This cueing effect gradually disappeared throughout the course of the experiment. In addition, participants performed at chance level in the explicit odor localization task, thus confirming the results of previous research.The implicit cueing task suggests the existence of spatial information processing in the olfactory system. Response slowing after a side-congruent olfactory cue is interpreted as a cross-modal attentional interference effect. In addition, habituation might have led to a gradual disappearance of the cueing effect. It is concluded that under immobile conditions with passive monorhinal stimulation, humans are unable to explicitly determine the location of a pure odorant. Implicitly, however, odor localization seems to exert an influence on human behaviour. To our knowledge, these data are the first to show implicit effects of odor localization on overt human behaviour and thus support the hypothesis of residual directional smelling in humans

Predictors and outcomes in primary depression care (POKAL) – a research training group develops an innovative approach to collaborative care

BACKGROUND: The interdisciplinary research training group (POKAL) aims to improve care for patients with depression and multimorbidity in primary care. POKAL includes nine projects within the framework of the Chronic Care Model (CCM). In addition, POKAL will train young (mental) health professionals in research competences within primary care settings. POKAL will address specific challenges in diagnosis (reliability of diagnosis, ignoring suicidal risks), in treatment (insufficient patient involvement, highly fragmented care and inappropriate long-time anti-depressive medication) and in implementation of innovations (insufficient guideline adherence, use of irrelevant patient outcomes, ignoring relevant context factors) in primary depression care. METHODS: In 2021 POKAL started with a first group of 16 trainees in general practice (GPs), pharmacy, psychology, public health, informatics, etc. The program is scheduled for at least 6 years, so a second group of trainees starting in 2024 will also have three years of research-time. Experienced principal investigators (PIs) supervise all trainees in their specific projects. All projects refer to the CCM and focus on the diagnostic, therapeutic, and implementation challenges. RESULTS: The first cohort of the POKAL research training group will develop and test new depression-specific diagnostics (hermeneutical strategies, predicting models, screening for suicidal ideation), treatment (primary-care based psycho-education, modulating factors in depression monitoring, strategies of de-prescribing) and implementation in primary care (guideline implementation, use of patient-assessed data, identification of relevant context factors). Based on those results the second cohort of trainees and their PIs will run two major trials to proof innovations in primary care-based a) diagnostics and b) treatment for depression. CONCLUSION: The research and training programme POKAL aims to provide appropriate approaches for depression diagnosis and treatment in primary care

Lessons learned from applying established cut-off values of questionnaires to detect somatic symptom disorders in primary care: a cross-sectional study

IntroductionBased on two diagnostic accuracy studies in high-prevalence settings, two distinctly different combinations of cut-off values have been recommended to identify persons at risk for somatic symptom disorder (SSD) with the combination of the Patient-Health Questionnaire-15 (PHQ-15) and the Somatic Symptom Disorder—B Criteria Scale (SSD-12). We investigated whether the reported sensitivity and specificity of both recommended cut-off combinations are transferable to primary care.MethodsIn a cross-sectional study, 420 unselected adult primary care patients completed PHQ-15 and SSD-12. Patients scoring ≥9 and ≥ 23 (recommended cut-off combination #1) or ≥ 8 and ≥ 13 (recommended cut-off combination #2) were considered test-positive for SSD, respectively. To assess the validity of the reported sensitivity and specificity in different low- to high-prevalence settings, we compared correspondingly expected proportions of test positives to the proportion observed in our sample.ResultsBased on combination #1, 38 participants (9%) were found to be test positive, far fewer than expected, based on the reported values for sensitivity and specificity (expected minimum frequency 30% with a true prevalence ≥1%). This can only be explained by a lower sensitivity and higher specificity in primary care. For combination #2, 98 participants (23%) were test positive, a finding consistent with a true prevalence of SSD of 15% or lower.DiscussionOur analyzes strongly suggest that the sensitivity and specificity estimates reported for combination #1 are not applicable to unselected primary care patients and that the cut-off for the SSD (≥23) is too strict. Cut-off combination #2 seems more applicable but still needs to be tested in studies that compare screening findings by questionnaires with validated diagnostic interviews as reference standards in primary care populations