Le virus Herpes Simplex de type 1 (HSV1) est responsable chez l’homme, son seul hôte naturel, d’infections oculaires cornéennes (kératites) récurrentes, typiquement unilatérales, pouvant induire une perte majeure de la vision. Pendant toute la vie, le virus reste à l’état quiescent (latence) dans le système nerveux, en particulier dans les deux ganglions trigéminés (TG) qui sont responsables de l’innervation sensitive de la cornée. La réactivation du virus à partir de ces TG entraine la kératite. Jusqu’à présent, les seuls traitements disponibles contre HSV1 ne sont que curatifs, c’est à dire qu’ils permettent de contrôler la réactivation que lorsque est déclarée. Il n’existe pour l’instant aucune thérapeutique réellement préventive sur l’ensemble des récidives, en particulier aucun vaccin n’a fait la preuve de son efficacité.Notre équipe a caractérisé un modèle d’infection herpétique par primo-infection orale, qui reproduit chez la souris une grande partie de l’histoire naturelle de l’infection herpétique telle qu’elle est observée chez l’homme. Ce modèle reproduit aussi la latéralisation, puisque la kératite initiale (puis ses récidives éventuelles) n’est observée que du côté inoculé, alors que l’infection latente est retrouvée dans les deux TG. Cependant, cette latence bilatérale n’est pas parfaitement symétrique à l’échelle moléculaire: alors que la charge virale latente (nombre de copies de génome) est similaire entre les deux TG, la production de LAT (Latency-Associated Transcripts) est plus importante du côté inoculé, de même que le nombre de neurones exprimant ces LAT (Cavallero et al., 2014; Maillet et al., 2006). Or, l’expression des LAT, marqueur classique de l’infection latente par HSV1, est associée, d’après la littérature scientifique, à la possibilité ultérieure de réactivation virale. A l’inverse, une infection herpétique sans expression des LAT est considérée comme peu réactivable (Perng et al., 2000). L’asymétrie biologique observée dans notre modèle pourrait donc expliquer la plus forte capacité de réactivation de HSV1 du côté inoculé seulement.L’objectif de l’ensemble de notre projet a été de tenter de contraindre une souche virale sauvage (à virulence normale) à une infection latente mais à capacité réduite de réactivation (sans expression de LAT), c’est à dire comme observé du côté non-inoculé de notre modèle. Pour cela, nous avons étudié l’effet de la primo-infection herpétique d’une souche de HSV1 sur la sensibilité des tissus à héberger l’infection latente par une autre souche virale, inoculée ultérieurement et dans un autre site.Notre avons montré que la primo-infection par une souche de HSV1 a inhibé la pathogénie (morbidité et mortalité) induite une autre souche de HSV1 virulente, inoculée quelques jours après. La primo-infection a contraint cette souche réinfectante à une mise en latence sans réplication au préalable, cette latence ne s’accompagnant pas d’expression de LAT. Cet effet inhibiteur a également été observé lors de l’utilisation d’une souche atténuée, non virulente dans le système nerveux, lors de la primo-infection. L’étude de la réactivation des différentes souches a révélé que lors de l’utilisation d’une souche neurovirulente et réactivable en tant que souche de primo-infection, la souche réinfectante pouvait également réactiver (aussi bien que son contrôle). En revanche, lors d’une primo-infection avec une souche non réactivable, la réactivation de la souche réinfectante était presque entièrement inhibée.La primo-infection par une souche non neurovirulente a contraint une souche, réellement virulente et inoculée secondairement, à une infection latente sans capacité de réactivation. Nous disposons ainsi des bases du développement d’une stratégie réellement préventive de l’infection herpétique récidivante, premier temps d’une éventuelle utilisation à des fins vaccinales. / The Herpes Simplex virus 1 (HSV1), whose only natural hosts are humans, can persist during the whole lifetime in a quiescent state (latent infection) in the nervous system, especially in both trigeminal ganglia (TGs, right and left), which innervate the cornea. The virus can reactivate in the TG, leading to recurrent corneal infections (keratitis) that are typically unilateral and can lead to major vision loss. To date, the only available therapies against HSV1 are curative, i.e. they control the reactivation process only after its onset. Until now, no efficient preventive treatment against HSV1 has been established, and more specifically no vaccine has been shown to be clinically effective.Our team has developed an oro-ocular murine model (based on viral inoculation in the lip), that mimics most of the aspects of the natural history of HSV1 infection in humans. In particular, lateralization is also found in this model, as only the eye ipsilateral to the inoculated lip develops keratitis (initial keratitis and recurrences), while latent virus is found in both TGs with similar levels of viral genome copies. However, the bilateral latency isn’t perfectly symmetrical at the molecular level, since the production of Latency-Associated Transcripts (LATs) and the number of LAT+ neurons are higher in the ipsilateral TG (Cavallero et al., 2014; Maillet et al., 2006). As LAT expression is associated with the capacity of the virus to reactivate, the asymmetry in LAT expression could explain the unilaterality of keratitis events.The aim of this project was to constraint a wild-type HSV1 strain to enter a non-reactivable state of latent infection in the both TGs. As this peculiar type of latent infection is observed only in the controlateral TG following a unilateral primary infection, we hypothesized that this phenomenon is linked to the kinetics of HSV1 infection in the both TGs, respectively. To test this, we studied the impact of a primary HSV1 infection on the behavior (acute phase, latency, LAT expression, capacity of reactivation) of a superinfecting HSV1 strain, inoculated at another anatomical site some days later.We have shown that the primary infection with a HSV1 strain can inhibit the pathogeny (morbidity and mortality) of a superinfecting virulent HSV1 strain, inoculated few days afterwards. Moreover, the superinfecting strain was found to be very rapidly driven in a latent state, with very poor LAT expression. This inhibitory effect also occurred when using a non-neurovirulent strain of HSV1 for the primary infection, with no further ability of the wild-type superinfecting strain to reactivate.These results clearly show that the onset of productive infection in the TGs and later on, latent infection with putative reactivation, is related to the kinetics of infection. These observations may have implications in the future for the potential development of innovative preventive strategies.

Approximately 10% of all gastric cancer cases worldwide are associated with Epstein-Barr virus (EBV) infection. This subgroup differs from non-infected equivalents through a distinct mutational and epigenetic signature, which is crucial for the establishment and maintenance of the viral latent state. Disruption of the latent state and subsequent induction of the lytic cycle, called reactivation, can be used as a therapeutic strategy for this cancer type through either combination with antiviral agents or by induction of an EBV-specific immune response. In this study, epigenetic inhibitors were tested for their potential to reactivate EBV using a novel screening method based on the expression of Zebra, a key regulator of EBV reactivation. Histone deacetylase inhibitors (HDACis), more specifically benzamide-based HDACis, were the most potent group of reactivating agents in this screen. Those HDACis induced the expression of lytic cycle genes as well as targeted cell killing in combination with antiviral agents. Moreover, the reactivation through those agents was sufficient to activate cytotoxic T-cells and thereby ensured a supportive role of the immune system in targeted cell killing. In an additional screen, topoisomerase inhibitors and other chemotherapeutic agents showed synergistic effects on Zebra expression together with HDACis, which was linked to the induction of p53 expression through those compounds. While p53 is known to be involved in the expression of Zebra, the viral DNA processivity factor Ea-D and the viral DNA binding protein DNBI were identified as a potential novel binding protein of p53 and could thus point towards a novel role of p53 in EBV lytic replication. Taken together, this study identifies potential novel therapeutic compound combinations for the treatment of EBV-associated epithelial cancers that could be translated into the clinic after further assessment.

Organophosphorus compounds (OPs) are used as pesticides, e.g. parathion, which is converted in the body to paraoxon, and chemical warfare nerve agents, such as sarin, soman, cyclosarin, VX, and tabun. Even small amounts of OP exposure can be fatal, depending on the toxicity of the compound. Great stocks of highly toxic chemical warfare nerve agents exit around the world and are considered a serious threat to national security and international stability. OPs exert their toxicity by covalent irreversible inhibition of acetylcholinesterase (AChE) that prevents the enzyme from hydrolyzing acetylcholine (ACh), a neurotransmitter in the central and peripheral nervous systems (CNS and PNS). Therefore, ACh accumulates in the cholinergic synapses throughout the body, which results in overstimulation of the ACh receptors. Removal of the phosphyl moiety from the OP-bound AChE active site has been a promising method to restore AChE’s catalytic activity. However, a secondary process called aging also occurs in the OP-AChE complex. Once aging occurs, currently available oximes are ineffective in removing the phosphyl moiety from the enzyme’s active site, and hence are ineffective as antidotes against the aged enzyme. Several families of alkylating and acylating agents including several classes of agents that combine alkylating moieties with known active site or peripheral cite (PAS) binding motifs were synthesized and evaluated. The general aim of the research was that successful alkylation or acylation of the phosphonate monoanion of aged AChE would produce neutral phosphyl complexes that would either spontaneously reactivate or would be reactivatable in the presence of oxime antidotes. Methoxylamine analogs of the oxime antidote 2-PAM were synthesized with the aim that methyl transfer to the aged AChE adduct would produce a neutral phosphyl AChE adduct simultaneously with 2-PAM in situ, and subsequent 2-PAM nucleophilic attack would reactivate the newly formed neutral phosphyl-AChE adduct. However, none of these 2-PAM analogs resurrected the activity of aged AChE. Another strategy for resurrecting the activity of aged AChE utilizes N-methylpyridiniums that are substituted at the 2-position with a beta-lactam moiety. For these compounds, opening of the electrophilic beta-lactam unmasks a nucleophilic amidine function which could putatively attack at phosphorus to expel the free enzyme. For this class of agents, only the active site directed compound that possessed the 5-CF₃ substituent showed possible resurrection of the activity of aged AChE, though activities in both the control and treated samples were low. Methyl transfers are common in Nature, and the natural transfer agent is S-adenosylmethionine, a sulfonium methyl donor. Consequently, the array of sulfonium compounds were evaluated on the expectation that they would bind to the AChE active site and transfer a methyl group to the phosphonate monoanion of the aged enzyme. Though high-affinity binding was noted for these compounds, none of these resurrected the activity of the aged AChE complex. Finally, several selected agents were evaluated on reactivating the initial OP-AChE complex before aging has occurred. It was observed that degraded samples of selected inhibitors are capable of reactivating initial complexes of sarin and soman inhibited AChE at low concentration that is an important character of efficient reactivators. However, the structure of reactivator is still unknown. Two major challenges still face researchers in the quest to design effective medicinal agents for counteracting poisoning by AChE-inhibiting nerve agents. The first is that there is no universal oxime antidote. Oximes that are effective against certain nerve agents are ineffective against others. The second is that, despite extensive efforts that span two generations, aged phosphyl-AChE adducts have never been reactivated. However, given the powerful tools of modern structural biology, medicinal chemistry and molecular biology, there is still hope that these considerable challenges can be met.

In human infection, herpes simplex virus (HSV) navigates two distinct life cycles; lytic and latent. The latent cycle takes place in sensory neurons, and is characterized as a dormant period punctuated by stress-induced episodes of viral reactivation. Understanding the mechanisms by which HSV latency and reactivation occur has been hindered by the lack of a model that faithfully recapitulates the environment of a human sensory neuron. Systems ranging from rat neurons to human fibroblasts have been developed to host HSV latency, however few available models have been able to investigate the role of human neuron-specific factors. To address this need, human medulloblastoma tumour cell lines, which derive from neuronal precursor cells, were differentiated and examined for their ability to host the HSV latency-reactivation cycle—in a manner similar to the differentiated PC-12 cell model. ONS-76 and UW228 medulloblastoma cell lines were screened for differentiation capacity. The differentiated cells were demonstrated to possess neuronal character as several neuron-specific proteins were found to be expressed. Differentiated ONS-76 cells were not compatible with hosting HSV latency, however, infection with a viral mutant impaired for lytic cycle initiation exhibited a deviant pattern of gene expression that resembles what has been observed in reactivation. Differentiated UW228 cells were found to host a low frequency, stable infection with the HSV mutant, characterized by the absence of infectious virus and viral lytic gene expression in the presence of persisting viral DNA. This DNA could further be induced to re-enter the lytic cycle through heat shock treatment and removal of differentiating agents from cell cultures. These results depict differentiated medulloblastoma cells as a novel tool in the study of HSV latency and reactivation, as these cells derive from the central nervous system and provide a new cellular perspective through which HSV biology can be viewed.

The post-war development of reunified Germany has resulted in many physical, economic, social and cultural changes. Despite the end of many restrictions imposed upon its populace during the Cold War, change would become an unexpected challenge to the people of a new Germany. With their residual memories from an extinct authoritative system, the general populace hinder redevelopment, and ultimately leave neighboring communities in a state of continued separation. The following thesis investigates the physical, social and cultural characteristics of site in attempts at generating, as an architectural methodology, infrastructural and programmatic strategies capable of informing the redevelopment of derelict post-industrial sites. In addressing the latent characteristics of site, historical, physical and programmatic, the resulting infrastructural and architectural framework assumes a programmatic classification that emphasizes its current dynamic uses and temporary programmes, enables changeability, and maintains memory of place by way of uninhibited openness for its users and surrounding communities.

The aim of this research was to determine whether a series of novel pyridinium oximes that were synthesized to incorporate phenoxyalkyl moieties to increase lipophilicity, and thereby increase their likelihood of crossing the blood-brain barrier, could effectively reactivate phosphorylated AChE in vitro. An experimental OP was synthesized as a surrogate for sarin to test the reactivation potential of the oximes, phthalimidyl isopropyl methylphosphonate (PIMP). The reactivation activities of the novel pyridinium oximes on PIMP exposed AChE and structure activity relationships were examined. Differences in reactivation potential in comparison to the widely used 2-PAM were also examined. All the novel oximes tested demonstrated some ability to reactivate inhibited AChE. Percent reactivations varied among the oximes (24%-78%), and the novel oximes were not as effective as 2-PAM or TMB-4, which reactivated 91 and 97%, respectively. The lipophilicity for all oximes was greater than 2-PAM or TMB-4 by 3- to 374- fold. A few of the novel oximes showed combined higher lipophilicity and reactivation potential approaching that of 2-PAM, and therefore suggest some potential efficacy as brain-penetrating oxime reactivators.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with exacerbation of clinical symptoms and induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive and latent HSV-1 and HSV-2 infection within sensory and autonomic neurons, we analyzed viral DNA after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal (TG) and sympathetic superior cervical ganglia (SCG) neurons expressed adrenergic receptors and glucocorticoid receptor. In productively infected neuronal cultures, epinephrine treatment significantly increased HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased HSV-2 DNA replication and production of viral progeny in SCG neurons, but not in TG neurons. In quiescently infected neuronal cultures, epinephrine and corticosterone significantly increased HSV-1 reactivation from sympathetic SCG neurons, but not sensory TG neurons. In contrast, corticosterone increased HSV-2 reactivation from both SCG and TG neurons, but epinephrine had no effect. Adrenergic or epinephrine-induced reactivation of HSV-1 in SCG neurons involved activation of several adrenergic receptors, the cyclic AMP response element binding protein (CREB), the transcription factor β-catenin, and the c-Jun N-terminal kinase (JNK). Corticosterone-induced reactivation of HSV-1 in SCG neurons required activation of glucocorticoid receptor (GCR) and transcription factors CREB and JNK. In contrast, corticosterone-induced reactivation of HSV-2 in TG and SCG neurons could utilize either the GCR or mineralocorticoid receptor (MCR) and most likely involves the chromatin remodeling properties of those receptors. Thus, stress-related hormones, epinephrine and corticosterone, selectively modulate productive and quiescent HSV-1 and HSV-2 infections primarily in sympathetic, but not sensory, neurons through different mechanisms. These results have implications for describing a mechanism by which stress-induced reactivation may occur in humans. / Ph. D. / Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2) are major human pathogens, which establish latency in neurons of the peripheral nervous system and reactivate to cause recurrent disease in humans. Physiological stress, which includes the secretion of the stress hormones epinephrine and cortisol, has been associated with increases in severity of clinical signs and increased recurrent disease in humans and animal models of herpetic disease. The mechanism by which physiological stress induces HSV reactivation has been assumed to be through suppression of the immune system. In addition, it has been assumed that sensory neurons harboring latent HSV are the primary source of reactivating virus for recurrent HSV disease. However, my dissertation provides evidence that the stress hormones epinephrine and corticosterone (the rodent equivalent of cortisol) can act on peripheral neurons in which the virus is latent, rather than through immune system suppression. In addition, my dissertation provides evidence that the autonomic nervous system, which modulates the physiological stress response, is an important source of reactivating virus to cause recurrent disease. The molecular pathway by which epinephrine and corticosterone induce HSV reactivation in primary adult murine neurons involves specific receptors, transcription factors, and protein kinases that could potentially be targeted in humans for inhibition of HSV reactivation and prevention of herpetic recurrent disease.

Patterns of hippocampal ensemble activity that occur during a spatial experience are reactivated during subsequent rest periods and slow wave sleep. Connections between active cells are thought to be strengthened, via long term potentiation (LTP), by repeated co-activation during experience, which suggests that the level of memory trace reactivation would increase proportionately with repetition. Alternatively, plasticity associated with memory formation, such as LTP-dependent place field expansion and the induction of activity-dependent immediate early gene, ARC, saturates after only a few laps, indicating that reactivation would plateau after a few repetitions. The length of the repeated sequence may also affect reactivation, since activation of a very short sequence can be repeated more frequently than a long sequence in a given time period. We studied how memory trace reactivation was affected by repetition and the length of the repeated sequence by observing the reactivated patterns of cell-pair correlations after a rat ran laps around a long circular track versus running more laps around a short track. On the shorter track, fewer cells had place fields, but they covered more of the track, resulting in generally stronger correlations among active cells. In addition, neuronal activity was recorded from dorsal and mid-ventral CA1. In mid-ventral CA1, there were fewer place fields in the environment but they were larger, with generally stronger correlations among active cells. The comparison between dorsal and mid-ventral regions is thus analogous to the comparison between the sequence of place fields on a long versus short track, respectively. Although there were more cells active in the dorsal region, but more potent correlations in the middle region, no differences in memory trace reactivation were found with respect to repetitions, track length or hippocampal region. This suggests that although spatial scaling increased along the dorsoventral axis of the hippocampus, reactivation is balanced, and possibly coherent across the hippocampal axis and it is relatively independent of sequence length or number of repetitions, at least when that number exceeds about 20.

Prior research shows that a contextual reminder can return a previously consolidated memory to an unstable state similar to initial encoding. New knowledge presented before the trace is reconsolidated can emerge as updating of the first experience with knowledge from the second. Sleep has been implicated in the long-term strengthening and storage of newly acquired episodic memories; thus, the delay-dependent emergence of intrusions may be facilitated by a delay containing sleep. The experiments described here explore this possibility by tracking sleep while participants undergo an episodic reconsolidation paradigm, which involves learning two sets of information and a recall task, all separated by 48 hours. Prior work using this paradigm shows that reminding participants of the first learning experience prior to learning the second renders them more likely to intrude information from the second set in their recall of the first. In the present study, Experiment 1 compares amount of sleep across days in order to tease apart the differential effects on consolidation of the original Set 1 memory and its reconsolidation when it is updated with Set 2. In Experiment 2, the first analysis (Analysis A) identifies events in the sleep EEG, such as spindles, that are associated with certain elements of memory consolidation, and expands on the parameters in which they occur in memory reconsolidation in Analysis B. The overall aim of this project is to use sleep as a means to inform the nature of memory reconsolidation, which paints memory as ever changeable.

Memory updating has been established; however, the mechanism supporting this alteration process is subject to disagreement. Reconsolidation theorists argue that memory updating occurs via an old memory becoming reactivated and returned to a state of susceptibility. In this state, newly presented details can become incorporated into the existing memory. As such, memory updating is an effect of old memory reactivation and new information encoding. In contrast, temporal context theory argues that the temporal context in which the old memory was initially formed is reinstated. Newly presented information becomes tagged to the old context. Therefore, at retrieval, when the old context is reinstated again, the initially bound information and the newer information are simultaneously retrieved. Within this theoretical framework, memory modification is the result of retrieval effects. In contrast, this three-paper dissertation provides evidence that reconsolidation is, at least in part, a combined reactivation and encoding effect. In paper 1, I present neural evidence of both old memory reactivation and new encoding, which demonstrates 1) that strength at reactivation predicts the likelihood that a memory will be modified and 2) that greater brain activation during new encoding predicts the extent of accurate recognition. In paper 2, I show that encoding conditions affect the extent to which new information will be misattributed to the old memory. I demonstrate that learners update explicitly encoded memories but not implicitly coded ones. Lastly, in paper 3, I demonstrate that old memories can be reactivated and altered during sleep. When old-memory reactivation is paired with a forget cue, a subsequent degeneration of the memory and its details ensues. In sum, all three papers provide evidence in support of the reconsolidation theory that memory updating occurs during old-memory reactivation and new encoding.