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When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions., SCOPUS: ar.j, FLWIN, info:eu-repo/semantics/published

Le virus de la rage est un pathogène neurotrope qui, actuellement, fait toujours plus de 55000 morts par an et dont la pathogenèse présente toujours de nombreuses zones d’ombre. Le mécanisme par lequel le virus échappe au système immunitaire de même que l’implication d’autres types cellulaires, particulièrement au niveau de la phase de latence, n’est pas encore connu. Il en est de même pour le mécanisme par lequel le virus induit un dysfonctionnement neuronal. Dans le cadre de ce projet de thèse, nous avons donc décidé d’étudier l’implication du macrophage et des caspases dans l’infection induite par le virus rabique. Nos résultats ont permis de démontrer que, malgré la faiblesse de la production virale in vitro, les macrophages infectés par une souche virulente de virus rabique étaient capables de transmettre l’infection et d’induire une encéphalite mortelle chez la souris. Ces résultats suggèrent que les macrophages sont susceptibles d’être des hôtes potentiels du virus.Dans un deuxième temps, nous avons également mis en évidence des différences de caractéristiques d’infection entre les souches virulentes et atténuées du virus rabique. Nous avons en effet démontré qu’en comparaison avec la souche vaccinale ERA, l’infection par la souche virulente CVS-11 était moins productive dans les cellules d’origine périphérique, tels que les cellules musculaires ou les macrophages. En revanche, la souche virulente se réplique mieux dans le cerveau et dans les cellules d’origine neuronale. Chez le macrophage, nous avons pu observer que l’infection par la souche atténuée ERA induisait une plus forte mortalité que celle générée par la souche CVS-11. Nos analyses ont mis en évidence que cette mortalité était de type apoptotique et qu’elle impliquait l’activation des caspases-1, -3, -7, -8, -9, du facteur Bid et de l’IL-1β. Des analyses réalisées sur des macrophages knock-out pour les caspase-1, -3 et -7 ont démontré d’une part l’importance de la caspase-3 dans la mortalité, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

For efficient prevention of viral infections and cross protection, simultaneous targeting of multiple viral epitopes is a powerful strategy. Llama heavy chain antibody fragments (VHH) against the trimeric envelope proteins of Respiratory Syncytial Virus (Fusion protein), Rabies virus (Glycoprotein) and H5N1 Influenza (Hemagglutinin 5) were selected from llama derived immune libraries by phage display. Neutralizing VHH recognizing different epitopes in the receptor binding sites on the spikes with affinities in the low nanomolar range were identified for all the three viruses by viral neutralization assays. By fusion of VHH with variable linker lengths, multimeric constructs were made that improved neutralization potencies up to 4,000-fold for RSV, 1,500-fold for Rabies virus and 75-fold for Influenza H5N1. The potencies of the VHH constructs were similar or better than best performing monoclonal antibodies. The cross protection capacity against different viral strains was also improved for all three viruses, both by multivalent (two or three identical VHH) and biparatopic (two different VHH) constructs. By combining a VHH neutralizing RSV subtype A, but not subtype B with a poorly neutralizing VHH with high affinity for subtype B, a biparatopic construct was made with low nanomolar neutralizing potency against both subtypes. Trivalent anti-H5N1 VHH neutralized both Influenza H5N1 clade1 and 2 in a pseudotype assay and was very potent in neutralizing the NIBRG-14 Influenza H5N1 strain with IC 50 of 9 picomolar. Bivalent and biparatopic constructs against Rabies virus cross neutralized both 10 different Genotype 1 strains and Genotype 5. The results show that multimerization of VHH fragments targeting multiple epitopes on a viral trimeric spike protein is a powerful tool for anti-viral therapy to achieve "best-in-class" and broader neutralization capacity. © 2011 Hultberg et al.

Influenza A virus infections impose a recurrent and global disease burden. Current antivirals against influenza are not always effective. Weassessed the protective potential ofmonovalent and bivalent Nanobodies (Ablynx) against challenge with this virus. These Nanobodies were derived from llamas and target H5N1 hemagglutinin. Intranasal administration of Nanobodies effectively controlled homologous influenza A virus replication. Administration of Nanobodies before challenge strongly reduced H5N1 virus replication in the lungs and protected mice from morbidity and mortality after a lethal challenge with H5N1 virus. The bivalent Nanobody was at least 60-fold more effective than the monovalent Nanobody in controlling virus replication. In addition, Nanobody therapy after challenge strongly reduced viral replication and significantly delayed time to death. Epitope mapping revealed that the VHH Nanobody binds to antigenic site B in H5 hemagglutinin. Because Nanobodies are small, stable, and simple to produce, they are a promising, novel therapeutic agent against influenza. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

Cancer is caused by the uncontrolled proliferation of aberrant cells that harbour multiple oncogenic mutations in signaling pathways implicated in growth and cell death. Most of today’s anticancer therapies lead to destruction of tumour cells by apoptosis. However, mutations leading to the development of cancer not only promote tumour progression, but often allow cells to avoid apoptotic death and thus result in resistance to treatment. Fortunately, many pathways lead to apoptosis and alternative cell death programmes do exist. Advances in the knowledge about how apoptosis and alternative cell death are activated are important and could pave the way for the development of alternative therapies. Using lymphoid cell lines, in particular Jurkat T cells, we have studied cell death pathways induced by an antiviral response or a prolonged endoplasmic reticulum (ER) stress. First, we have studied the involvement of RIP1 and the activation of dsRNA-activated protein kinase (PKR) in external dsRNA-induced apoptotic and necrotic cell death in Jurkat T cell lymphoma. Our findings suggest that RIP1 is implicated in apoptosis and necrosis induced by dsRNA and a caspase-dependent mode of activation of PKR in apoptosis. PKR is cleaved in two fragments, PKR-ND containing the N-terminal domain of PKR and PKR-KD containing the kinase domain. The PKR-KD fragment interacts with and activates intact PKR. PKR-ND facilitates the interaction of PKR-KD with full-length PKR and thus the activation of the kinase and amplifies the translation inhibitory signal. Secondly, we explored the pathways leading to caspase activation and the involvement of Bcl2 pro-apoptotic members and RIP1 in cell death induced by ER stress in Jurkat T cells. Our results show that caspases-3, -8 and -9 are implicated in apoptosis induced by brefeldin-A (BFA). Using caspase-8 and FADD deficient Jurkat T cells, we demonstrate that these proteins are redundant for BFA induced apoptosis. In Bcl2 overexpressing Jurkat cel, Le cancer est causé par la prolifération incontrôlée de cellules suite à de nombreuses altérations génétiques affectant notamment les voies de signalisation impliquées dans la croissance et la mort cellulaires. La majorité des thérapies anticancéreuses visent à induire l’apoptose des cellules cancéreuses, cependant, les cellules y deviennent de plus en plus résistantes. Heureusement, le concept d’apoptose ne représente pas le seul mécanisme pouvant mener à la mort cellulaire et d’autres types de mort émergent et doivent être pris en considération. Une meilleure compréhension des mécanismes d’apoptose et des modes alternatifs de mort cellulaire permettrait de déterminer de nouvelles cibles dans le but d’améliorer certaines thérapies actuelles. Nos recherches ont porté sur l’étude de la mort cellulaire induite lors d’une réponse antivirale ou lors d’un stress du réticulum endoplasmique (RE) dans des lignées lymphoïdes et plus particulièrement, les cellules T Jurkat. D’une part, nous nous sommes intéressés à décortiquer les mécanismes de l’activation de la kinase PKR (double stranded RNA-dependent kinase) par les caspases et à l’implication de RIP1 au cours de l’apoptose et de la nécrose induites par l’ARNdb (ARN double brin) dans les cellules T Jurkat. Nos résultats suggèrent que RIP1 est impliqué dans l’apoptose et la nécrose induite par l’ARNdb. Nous mettons aussi en évidence un nouveau mode d’activation de PKR dépendant des caspases au cours de l’apoptose. La PKR est clivée par les caspases en fragments N-terminal (PKR-ND) et C-terminal porteur du domaine kinase (PKR-KD). Ces deux fragments montrent des fonctions différentes. PKR-KD est capable de se lier et d’activer la PKR tandis que PKR-ND amplifie ce processus en facilitant l’interaction entre PKR-KD et la kinase PKR entière. D’autre part, nous avons étudié l’implication des caspases, des membres de la famille Bcl-2 et de RIP1 (receptor-interacting protein-1) dans l’apoptose et la nécrose induites par un stress, (DOCSC03) -- FUNDP, 2010