Altération de la fluence verbale et de la flexibilité mentale chez des patients dépressifs unipolaires et bipolaires

La dépression est un problème majeur en Santé Publique et elle a un impact fonctionnel majeur sur la vie des personnes qui en sont atteintes. Elle entraine une détérioration cognitive sur les fonctions de mémoire, d’attention, exécutives et de rapidité d’action. <br>Dans ce travail, nous avons mesuré les fonctions exécutives, plus spécifiquement la flexibilité mentale et la fluence verbale au travers d’une étude comparative de 24 patients dépressifs (12 unipolaires, 12 bipolaires) et de 24 témoins (appariés sur le sexe, l’âge, et le niveau d’études). Nous avons comparé les performances en fluences verbales et graphiques, et en flexibilité mentale entre le groupe de patients dépressifs et le groupe de témoins, puis entre le groupe de dépressifs unipolaires et le groupe de dépressifs bipolaires. La valence affective à partir de la production en fluence verbale a été également étudiée. Nous avons retrouvé des performances significativement moindres dans le groupe des patients dépressifs et plus particulièrement dans le sous-groupe des bipolaires (en particulier au TMT-A, en fluence verbale et en fluence graphique). Ceci pourrait participer aux difficultés de langage et de communication retrouvées chez les patients dépressifs. Enfin, nous nous sommes intéressé à l’aspect psychopathologique et phénoménologique des dépressions, afin d’essayer de comprendre les baisses de performances des patients qui en souffrent, notamment en matière de langage, de communication, ou de flexibilité mentale

Characterisation of the nuclear targeting mechanisms, nuclear functions, and nuclear interactors of rabies virus P3 protein

Rabies virus (RABV) causes more than 55,000 human deaths worldwide annually [1, 2]. This is largely because current inactivated vaccines for protection against RABV infection are costly and difficult to administer, and as such not available to the majority of people that are at risk of infection [3-5]. Further, there are no effective treatments for symptomatic RABV infection. Thus, there is a serious need for development of improved vaccines, including safe live vaccines, as well as antiviral drugs. This requires determining the molecular mechanisms underlying viral infection and pathogenicity in detail, and characterising viral protein interactions with the host cell. Among other recent studies that address these aims [6-9], one important publication has demonstrated that regulated nucleocytoplasmic shuttling of the RABV phosphoprotein (P) is essential for viral pathogenicity, as RABV expressing P impaired for nucleocytoplasmic trafficking becomes sensitive to interferon-mediated immunity, and thus, highly attenuated in vivo [10, 11]. This data strongly implies that regulated nucleocytoplasmic trafficking of P represents a potential target for attenuating mutations and development of antiviral therapeutics. RABV P is expressed as a full length protein, P1, and four N-terminally truncated isoforms, P2-P5 [12]. The expression of multiple isoforms from the single P gene is thought to facilitate RABV P serving its multiple functions in viral infection, including as the non-catalytic viral polymerase cofactor that is required for replication, and as an antagonist of the host antiviral immune responses [13-16]. Intriguingly, although RABV replicates in the cytoplasm and the P1 and P2 isoforms are strongly cytoplasmic, P3-P5 are reported to localise mostly in the nucleus [17]. Strongly cytoplasmic localisation of P1 and P2 is widely accepted to be due to the activity of a nuclear export signal (NES) in the N-terminal region of P, while P3-P5, which lack key residues of this NES, are reported to be driven into the nucleus by a nuclear localisation signal (NLS) within the C-terminal domain (CTD) [17]. Importantly however, much remains unresolved about these trafficking signals including mechanisms involved in their regulation and host factors involved in their recognition, and this relatively simple model of P isoform nucleocytoplasmic trafficking has been called into question by subsequent studies which have identified several additional signals that modulate P nucleocytoplasmic shuttling [18, 19]. Further, while several P isoforms are reported to localise to the nucleus, very little is known regarding their specific intranuclear functions and interactions. This PhD project addressed many of these unknowns by analysing the precise sequences and mechanisms involved in nuclear trafficking of P protein isoforms, in particular the strongly nuclear P3, and investigating the nuclear functions and binding partners of this isoform. Quantitative analysis of the subcellular distribution of the five P protein isoforms led to the identification of a novel nuclear localisation signal in the N-terminal region of P3 (the N-NLS) which is recognised by the cellular importin (IMP) α/β heterodimer and requires the presence of P residues K54 and R55 . These key residues are truncated in P4 and P5, which consequently lack N-NLS activity and are diffusely localised throughout the nucleus and cytoplasm. P3 is thus the only strongly nuclear P protein isoform. Further characterisation revealed that activation of the N-NLS also requires truncation of residues P1-52, such that it is not active in P1, but only active in the context of P3. Importantly, P residues 1-52 also contain the NES that drives P1 and P2 into the cytoplasm, which thus overlaps with the N-NLS. To our knowledge, this is the first report of a novel type of trafficking module wherein the activity of two opposing targeting signals are co-regulated through truncation. This study thus redefines the model of P protein nuclear trafficking, revealing a highly efficient regulatory mechanism that drives strong and specific nuclear targeting of P3, consistent with the idea that P3 serves key intranuclear roles in infection. The exact nuclear functions of P3 have remained unknown, however previous reports have suggested that RABV and related viruses such as Hendra virus (HeV) and Nipah virus (NiV) target proteins to the nucleus in order to inhibit IFN signalling by antagonising intranuclear STAT1 [20-23]. This was tested directly for the first time in this study using P3 hindered for nuclear trafficking by mutation of key N-NLS residues. The data indicated that P3 nuclear localisation is not critical for its antagonism of STAT1, as both wild type P3 and P3 containing mutations to key N-NLS residues can physically interact with STAT1 and inhibit STAT1 signalling to similar extents. To determine genuine reasons for the strong and specific nuclear targeting of P3, the intranuclear distribution of P3 was examined in greater detail, and this led to the finding that P3 can colocalise with nucleoli, interact specifically with the core nucleolar protein, nucleolin (NCL), and moreover, that NCL is critically required for production of infective RABV particles. To our knowledge, this is the first demonstration of a rhabdovirus targeting the nucleolus and interacting with NCL, and makes RABV one of only a handful of negative stranded RNA viruses known to interact with nucleoli and nucleolar factors. Further analysis indicated that in addition to targeting nucleoli, P3 may target distinct sub-nuclear structures, and serves additional nuclear functions including inhibition of cellular RNA synthesis. The identification if a novel host-virus interaction within the nucleus and of potentially important nuclear functions for P3 is consistent with the premise that nuclear trafficking of P is a promising target for attenuating mutations. Together, the findings of this study indicate that nuclear trafficking of P3 serves critical roles in infection, likely by enabling specific modulation of nuclear and/or nucleolar functions by the virus within infected cells. These data have thus made significant contributions to our understanding of RABV biology, and identified the nucleolus as a new host-virus interface. Identifying host virus interactions, determining where they take place, and characterising the mechanisms and sequences involved is an extremely important prerequisite to develop new targets for attenuation, generate new vaccine strains, and design antiviral drugs against the currently incurable and fatal pathogen RABV, as well as related viruses

Characterisation of the nuclear targeting mechanisms, nuclear functions, and nuclear interactors of rabies virus P3 protein

Rabies virus (RABV) causes more than 55,000 human deaths worldwide annually [1, 2]. This is largely because current inactivated vaccines for protection against RABV infection are costly and difficult to administer, and as such not available to the majority of people that are at risk of infection [3-5]. Further, there are no effective treatments for symptomatic RABV infection. Thus, there is a serious need for development of improved vaccines, including safe live vaccines, as well as antiviral drugs. This requires determining the molecular mechanisms underlying viral infection and pathogenicity in detail, and characterising viral protein interactions with the host cell. Among other recent studies that address these aims [6-9], one important publication has demonstrated that regulated nucleocytoplasmic shuttling of the RABV phosphoprotein (P) is essential for viral pathogenicity, as RABV expressing P impaired for nucleocytoplasmic trafficking becomes sensitive to interferon-mediated immunity, and thus, highly attenuated in vivo [10, 11]. This data strongly implies that regulated nucleocytoplasmic trafficking of P represents a potential target for attenuating mutations and development of antiviral therapeutics. RABV P is expressed as a full length protein, P1, and four N-terminally truncated isoforms, P2-P5 [12]. The expression of multiple isoforms from the single P gene is thought to facilitate RABV P serving its multiple functions in viral infection, including as the non-catalytic viral polymerase cofactor that is required for replication, and as an antagonist of the host antiviral immune responses [13-16]. Intriguingly, although RABV replicates in the cytoplasm and the P1 and P2 isoforms are strongly cytoplasmic, P3-P5 are reported to localise mostly in the nucleus [17]. Strongly cytoplasmic localisation of P1 and P2 is widely accepted to be due to the activity of a nuclear export signal (NES) in the N-terminal region of P, while P3-P5, which lack key residues of this NES, are reported to be driven into the nucleus by a nuclear localisation signal (NLS) within the C-terminal domain (CTD) [17]. Importantly however, much remains unresolved about these trafficking signals including mechanisms involved in their regulation and host factors involved in their recognition, and this relatively simple model of P isoform nucleocytoplasmic trafficking has been called into question by subsequent studies which have identified several additional signals that modulate P nucleocytoplasmic shuttling [18, 19]. Further, while several P isoforms are reported to localise to the nucleus, very little is known regarding their specific intranuclear functions and interactions. This PhD project addressed many of these unknowns by analysing the precise sequences and mechanisms involved in nuclear trafficking of P protein isoforms, in particular the strongly nuclear P3, and investigating the nuclear functions and binding partners of this isoform. Quantitative analysis of the subcellular distribution of the five P protein isoforms led to the identification of a novel nuclear localisation signal in the N-terminal region of P3 (the N-NLS) which is recognised by the cellular importin (IMP) α/β heterodimer and requires the presence of P residues K54 and R55 . These key residues are truncated in P4 and P5, which consequently lack N-NLS activity and are diffusely localised throughout the nucleus and cytoplasm. P3 is thus the only strongly nuclear P protein isoform. Further characterisation revealed that activation of the N-NLS also requires truncation of residues P1-52, such that it is not active in P1, but only active in the context of P3. Importantly, P residues 1-52 also contain the NES that drives P1 and P2 into the cytoplasm, which thus overlaps with the N-NLS. To our knowledge, this is the first report of a novel type of trafficking module wherein the activity of two opposing targeting signals are co-regulated through truncation. This study thus redefines the model of P protein nuclear trafficking, revealing a highly efficient regulatory mechanism that drives strong and specific nuclear targeting of P3, consistent with the idea that P3 serves key intranuclear roles in infection. The exact nuclear functions of P3 have remained unknown, however previous reports have suggested that RABV and related viruses such as Hendra virus (HeV) and Nipah virus (NiV) target proteins to the nucleus in order to inhibit IFN signalling by antagonising intranuclear STAT1 [20-23]. This was tested directly for the first time in this study using P3 hindered for nuclear trafficking by mutation of key N-NLS residues. The data indicated that P3 nuclear localisation is not critical for its antagonism of STAT1, as both wild type P3 and P3 containing mutations to key N-NLS residues can physically interact with STAT1 and inhibit STAT1 signalling to similar extents. To determine genuine reasons for the strong and specific nuclear targeting of P3, the intranuclear distribution of P3 was examined in greater detail, and this led to the finding that P3 can colocalise with nucleoli, interact specifically with the core nucleolar protein, nucleolin (NCL), and moreover, that NCL is critically required for production of infective RABV particles. To our knowledge, this is the first demonstration of a rhabdovirus targeting the nucleolus and interacting with NCL, and makes RABV one of only a handful of negative stranded RNA viruses known to interact with nucleoli and nucleolar factors. Further analysis indicated that in addition to targeting nucleoli, P3 may target distinct sub-nuclear structures, and serves additional nuclear functions including inhibition of cellular RNA synthesis. The identification if a novel host-virus interaction within the nucleus and of potentially important nuclear functions for P3 is consistent with the premise that nuclear trafficking of P is a promising target for attenuating mutations. Together, the findings of this study indicate that nuclear trafficking of P3 serves critical roles in infection, likely by enabling specific modulation of nuclear and/or nucleolar functions by the virus within infected cells. These data have thus made significant contributions to our understanding of RABV biology, and identified the nucleolus as a new host-virus interface. Identifying host virus interactions, determining where they take place, and characterising the mechanisms and sequences involved is an extremely important prerequisite to develop new targets for attenuation, generate new vaccine strains, and design antiviral drugs against the currently incurable and fatal pathogen RABV, as well as related viruses

Subcellular trafficking in rhabdovirus infection and immune evasion: a novel target for therapeutics.

Vesicular stomatitis virus (VSV) and Rabies Virus (RABV) are the prototypic members of the rhabdovirus family. These viruses have a particularly broad host range, and despite the availability of vaccines, RABV still causes more than 50,000 human deaths a year. Trafficking of the virion or viral particles is important at several stages of the replicative life cycle, including cellular entry, localization into the cytoplasmic inclusion bodies which primarily house the transcription and replication of the viral genome, and migration to the plasma membrane from whence the virus is released by budding. Intriguingly, specific viral proteins, VSV M and RABV P have also been shown to undergo intracellular trafficking independent of the other viral apparatus. These proteins are multifunctional, and play roles in antagonism of host processes, namely the IFN system, and as such enable viral evasion of the innate cellular antiviral response. A body of recent research has been aimed at characterizing the mechanisms by which these proteins are able to shuttle between and localize to various subcellular sites, including the nucleus, which is not required during the cytoplasmic replicative life cycle of the virus. This work has indicated that trafficking of these proteins plays a significant role in determining the ability of the viruses to replicate and cause infection, and as such, represents a viable target for development of a new generation of vaccines and prophylactic therapeutics which are required to battle these pathogens of human and agricultural significance

Effects of hemostatic agents on shear bond strength of orthodontic brackets

Objectives: The aim of this study was to determine the effects of blood contamination and hemostatic agents on shear bond strength (SBS) of brackets and bond failure.Materials and Methods: The study material consisted of 57 freshly extracted human premolar and randomly divided into four groups: Group I, control group (n = 14); Group II, contamination with blood (n = 13); Group III, contamination with epinephrine (n = 14); and Group IV, contamination with Ankaferd blood stopper (ABS) (n = 16). After the bracket bonding procedure, all bonded teeth thermal cycled in deionized water at 5 ± 2°C to 55 ± 2°C for 500 cycles. SBS was applied using a universal test machine.Results: According to Kruskal–Wallis test significant differences were found among the groups P &lt; 0.05. Furthermore, significant differences were recorded between groups with Mann–Whitney U statistical test with Bonferroni correction (P = 0.0083).Conclusions: Examples contaminated with blood showed a statistically significant lower in vitro SBS than those contaminated with epinephrine, ABS, and control groups.Clinical Significance: In impacted tooth surgical operations, blood contamination poses a substantial risk of bond failure in bonding attachments applications to the impacted teeth. Epinephrine and ABS may be used on surgical exposed impacted teeth operation for the prevention of blood contamination.Key words: Brackets, contamination, hemostatic agents, orthodontic bonding, shear bond strengt