Les fluctuations du taux de calcium (Ca2+) intracellulaire sont impliquées dans les événements de signalisation et de régulation de différents processus cellulaires. Alors que le role du Ca2+ dans la réponse des plantes lors des interactions mycorhiziennes à arbuscules (MA) interactions est bien documentée, il n’existe aucune information concernant la régulation ou le rôle de ce messager secondaire chez le symbiote fongique. La base moléculaire de l'homéostasie calcique fongique dans la symbiose MA a été analysée en étudiant l'expression de gènes fongiques liés au Ca2+. Dans un premier temps, des gènes de G. mosseae codant putativement pour une protéine kinase-like MAP3k (Gm2) et une P-type ATPase (Gm152) ont été étudiés. L’expression des deux gènes est stimulée par les exudats racinaires d’A. sinicum, suggérant un rôle dans les interactions précoces avant l'établissement de la symbiose. L’obtention de la séquence d'ADNc pleine longueur de Gm152 a confirmé son identité. Une étude plus approfondie du rôle de Ca2+ dans les processus fongiques impliqués dans la symbiose MA a été réalisée chez G. intraradices. L'expression de sept gènes fongiques encodant six protéines de transport membranaire calcique et une protéine kinase nucléaire, sélectionnés du séquençage transcriptomique du G. intraradices, était stimulée lors de la colonisation des racines de M. truncatula type sauvage (lignée J5) mais pas chez le mutant non-mycorhizienne dmi3/Mtsym13. La cartographie par microdissection laser des transcrits des gènes fongiques a indiqué une activation différentielle dans les arbuscules et/ou dans hyphes intercellulaires. Les variations tempo-spatiales de l'expression des gènes fongiques suggèrent des roles différents dans le développement ou le fonctionnement de la symbiose MA. L’ADNc pleine longueur a été obtenue de trois gènes de G. intraradices encodant un PMR1-like réticulum endoplasmique ATPase, un VCX1-like transporteur ionique vacuolaire et un CCaMK nucléaire pour des analyses fonctionnelles chez la levure afin de mieux comprendre leur rôle dans la symbiose MA. Les mécanismes par lesquels les protéines liées au Ca2+ pourraient jouer un rôle chez G. intraradices dans la mobilisation et la perception du messager secondaire au cours des interactions MA sont discutés / Fluctuations in intracellular (Ca2+) calcium levels generate signaling events and regulate different cellular processes. Whilst the implication of Ca2+ in plant cell responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary meesenger in the fungal symbiont. The molecular basis of fungal calcium homeostasis in the AM symbiosis was analyzed by investigating the expression of Ca2+-related fungal genes. In a first study, G. mosseae genes putatively encoding a MAP3k-like protein kinase (Gm2) and a P-type ATPase (Gm152) were investigated. Both Ca2+-related genes were up-regulated by A. sinicum root exudates, suggesting a role in early interactions prior to symbiosis establishment. The full-length cDNA sequence of Gm152 obtained from germinating spores of G. mosseae confirmed its identity. The role of Ca2+ in fungal processes leading to establishment of an AM symbiosis was investigated in more detail in G. intraradices-M. truncatula interactions. Enhanced expression of genes encoding six membrane transport proteins and one nuclear protein kinase, selected from the G. intraradices transcriptome database, was related to colonization of wild-type M. truncatula (line J5) roots and not observed with the mycorrhiza-resistant mutant dmi3/Mtsym13. Laser microdissection mapping of transcripts indicated that the Ca2+-related G. intraradices genes were differentially up-regulated in arbuscules and/or in intercellular hyphae. The tempo-spatial variations in fungal gene expression suggest different roles in the development or functioning of the AM symbiosis. Full-length cDNA of three G. intraradices genes putatively encoding a PMR-like endoplasmic reticulum P-type ATPase, a VCX1-like vacuolar Ca2+ ion transporter and a nuclear CCaMK were obtained for functional analyses in yeast mutants to gain insight into their role in the mycorrhizal symbiosis. Possible mechanisms are discussed in which Ca2+-related proteins of G. intraradices may play a role in the mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots

Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...

Plasmacytoid dendritic cells (pDC) are a highly specialized subset of immune cells that sense viral nucleic acids by endosomal toll-like receptors 7 and 9 (TLR7/9). Activation of TLR7/9 leads to the production of type I interferons (IFN-I). Moreover, pDC contribute to the antiviral response by presenting viral antigens to T lymphocytes and link innate and adaptive immunity. pDC need to be properly regulated in order to limit excessive production of IFN-I that is associated with autoimmune diseases. Therefore, pDC possess a battery of regulatory receptors (RR) that limit TLR7/9-mediated cytokine production. This thesis focuses on the mechanism of RR-mediated inhibition of IFN-I production in pDC and explores interactions between pDC and two enveloped viruses, that possess the ability to hijack RR in pDC: hepatitis B virus (HBV) and human immunodeficiency virus (HIV). We showed, that MEK-ERK signaling pathway plays an active role in RR-mediated inhibition of IFN-I in pDC. Our results indicate that in line with other studies of our group, pharmacological targeting of MEK1/2-ERK signaling could be a strategy to re-establish immunogenic activity of pDC. Then, we investigated whether antiretroviral therapy (ART) in a cohort of 21 treatment-naive chronic HIV-infected patients has restored the number and...

<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>

Plasmacytoid dendritic cells (pDC) represent innate immune cells capable to detect viruses in their endosomal environment via Toll-like receptors (TLRs). Viral nuclear acid recognition leads to the massive production of type I interferon (IFN I) and induction of the antiviral state in uninfected cells. Crosslinking of the surface regulatory receptors, such as BDCA-2, with monoclonal antibodies or with some viruses leads to the activation of MEK1/2- ERK signaling pathway and inhibition of IFN I production in pDC. In this study, the role of MEK1/2 kinase has been highlighted. Its inhibition reversed the inhibitory effect of BDCA-2 crosslinking and its direct activation with PMA led to the inhibition of IFN-α production. Yet an unclear role of pDC in sensing of BK polyomavirus virus (BKV) responsible for kidney transplant rejection was investigated as a major topic of this thesis. Experiments with the pDC cell line Gen2.2 and HRPTEC primary cell line showed that pDCs were not able to detect BKV particles, however, exposure of activated Gen2.2 cells to BKV inoculum dramatically upregulated production of IFN-α. Most importantly, coculture of Gen2.2 cells with BKV- infected HRPTEC cells resulted in IFN-α and TNF-α production, which was prevented by Bafilomycin. These results suggest that BKV-infected...