Spontaneous Cdc42 polarization independent of GDI-mediated extraction and actin-based trafficking.

The small Rho-family GTPase Cdc42 is critical for cell polarization and polarizes spontaneously in absence of upstream spatial cues. Spontaneous polarization is thought to require dynamic Cdc42 recycling through Guanine nucleotide Dissociation Inhibitor (GDI)-mediated membrane extraction and vesicle trafficking. Here, we describe a functional fluorescent Cdc42 allele in fission yeast, which demonstrates Cdc42 dynamics and polarization independent of these pathways. Furthermore, an engineered Cdc42 allele targeted to the membrane independently of these recycling pathways by an amphipathic helix is viable and polarizes spontaneously to multiple sites in fission and budding yeasts. We show that Cdc42 is highly mobile at the membrane and accumulates at sites of activity, where it displays slower mobility. By contrast, a near-immobile transmembrane domain-containing Cdc42 allele supports viability and polarized activity, but does not accumulate at sites of activity. We propose that Cdc42 activation, enhanced by positive feedback, leads to its local accumulation by capture of fast-diffusing inactive molecules.

RNA interference is ineffective as a routine method for gene silencing in chick embryos as monitored by fgf8 silencing.

The in vivo accessibility of the chick embryo makes it a favoured model system for experimental developmental biology. Although the range of available techniques now extends to miss-expression of genes through in ovo electroporation, it remains difficult to knock out individual gene expression. Recently, the possibility of silencing gene expression by RNAi in chick embryos has been reported. However, published studies show only discrete quantitative differences in the expression of the endogenous targeted genes and unclear morphological alterations. To elucidate whether the tools currently available are adequate to silence gene expression sufficiently to produce a clear and specific null-like mutant phenotype, we have performed several experiments with different molecules that trigger RNAi: dsRNA, siRNA, and shRNA produced from a plasmid coexpressing green fluorescent protein as an internal marker. Focussing on fgf8 expression in the developing isthmus, we show that no morphological defects are observed, and that fgf8 expression is neither silenced in embryos microinjected with dsRNA nor in embryos microinjected and electroporated with a pool of siRNAs. Moreover, fgf8 expression was not significantly silenced in most isthmic cells transformed with a plasmid producing engineered shRNAs to fgf8. We also show that siRNA molecules do not spread significantly from cell to cell as reported for invertebrates, suggesting the existence of molecular differences between different model systems that may explain the different responses to RNAi. Although our results are basically in agreement with previously reported studies, we suggest, in contrast to them, that with currently available tools and techniques the number of cells in which fgf8 gene expression is decreased, if any, is not sufficient to generate a detectable mutant phenotype, thus making RNAi useless as a routine method for functional gene analysis in chick embryos.

Three-step pathway engineering results in more incidence rate and higher emission of nerolidol and improved attraction of Diadegma semiclausum

The concentration and ratio of terpenoids in the headspace volatile blend of plants have a fundamental role in the communication of plants and insects. The sesquiterpene (E)-nerolidol is one of the important volatiles with effect on beneficial carnivores for biologic pest management in the field. To optimize de novo biosynthesis and reliable and uniform emission of (E)-nerolidol, we engineered different steps of the (E)-nerolidol biosynthesis pathway in Arabidopsis thaliana. Introduction of a mitochondrial nerolidol synthase gene mediates de novo emission of (E)-nerolidol and linalool. Co-expression of the mitochondrial FPS1 and cytosolic HMGR1 increased the number of emitting transgenic plants (incidence rate) and the emission rate of both volatiles. No association between the emission rate of transgenic volatiles and their growth inhibitory effect could be established. (E)-Nerolidol was to a large extent metabolized to non-volatile conjugates.

Optimising approaches to active immunotherapy of cancer

The design of therapeutic cancer vaccines is aimed at inducing high numbers and potent T cells that are able to target and eradicate malignant cells. This calls for close collaboration between cells of the innate immune system, in particular dendritic cells (DCs), and cells of the adaptive immune system, notably CD4+ helper T cells and CD8+ cytotoxic T cells. Therapeutic vaccines are aided by adjuvants, which can be, for example, Toll¬like Receptor agonists or agents promoting the cytosolic delivery of antigens, among others. Vaccination with long synthetic peptides (LSPs) is a promising strategy, as the requirement for their intracellular processing will mainly target LSPs to professional antigen presenting cells (APCs), hence avoiding the immune tolerance elicited by the presentation of antigens by non-professional APCs. The unique property of antigen cross-processing and cross-presentation activity by DCs plays an important role in eliciting antitumour immunity given that antigens from engulfed dead tumour cells require this distinct biological process to be processed and presented to CD8+T cells in the context of MHC class I molecules. DCs expressing the XCR1 chemokine receptor are characterised by their superior capability of antigen cross- presentation and priming of highly cytotoxic T lymphocyte (CTL) responses. Recently, XCR1 was found to be also expressed in tissue-residents DCs in humans, with a simitar transcriptional profile to that of cross- presenting murine DCs. This shed light into the value of harnessing this subtype of XCR1+ cross-presenting DCs for therapeutic vaccination of cancer. In this study, we explored ways of adjuvanting and optimising LSP therapeutic vaccinations by the use, in Part I, of the XCLl chemokine that selectively binds to the XCR1 receptor, as a mean to target antigen to the cross-presenting XCR1+ DCs; and in Part II, by the inclusion of Q.S21 in the LSP vaccine formulation, a saponin with adjuvant activity, as well as the ability to promote cytosolic delivery of LSP antigens due to its intrinsic cell membrane insertion activity. In Part I, we designed and produced XCLl-(OVA LSP)-Fc fusion proteins, and showed that their binding to XCR1+ DCs mediate their chemoattraction. In addition, therapeutic vaccinations adjuvanted with XCLl-(OVA LSP)-Fc fusion proteins significantly enhanced the OVA-specific CD8+ T cell response, and led to complete tumour regression in the EL4-OVA model, and significant control of tumour growth in the B16.0VA tumour model. With the aim to optimise the co-delivery of LSP antigen and XCLl to skin-draining lymph nodes we also tested immunisations using nanoparticle (NP)-conjugated OVA LSP in the presence or absence of XCLl chemokine. The NP-mediated delivery of LSP potentiated the CTL response seen in the blood of vaccinated mice, and NP-OVA LSP vaccine in the presence of XCLl led to higher blood frequencies of OVA-specific memory-precursor effector cells. Nevertheless, in these settings, the addition XCLl to NP-OVA LSP vaccine formulation did not increase its antitumour therapeutic effect. In the Part II, we assessed in HLA-A2/DR1 mice the immunogenicity of the Melan-AA27L LSP or the Melan-A26. 35 AA27l short synthetic peptide (SSP) used in conjunction with the saponin adjuvant QS21, aiming to identify a potent adjuvant formulation that elicits a quantitatively and qualitatively strong immune response to tumour antigens. We showed a high CTL immune response elicited by the use of Melan-A LSP or SSP with QS21, which both exerted similar killing capacity upon in vivo transfer of target cells expressing the Melan-A peptide in the context of HLA-A2 molecules. However, the response generated by the LSP immunisation comprised higher percentages of CD8+T cells of the central memory phenotype (CD44hl CD62L+ and CCR7+ CD62L+) than those of SSP immunisation, and most importantly, the strong LSP+QS21 response was strictly CD4+T cell-dependent, as shown upon CD4 T cell depletion. Altogether, these results suggest that both XCLl and QS21 may enhance the ability of LSP to prime CD8 specific T cell responses, and promote a long-term memory response. Therefore, these observations may have important implications for the design of protein or LSP-based cancer vaccines for specific immunotherapy of cancer -- Les vacans thérapeutiques contre le cancer visent à induire une forte et durable réponse immunitaire contre des cellules cancéreuses résiduelles. Cette réponse requiert la collaboration entre le système immunitaire inné, en particulier les cellules dendrites (DCs), et le système immunitaire adaptatif, en l'occurrence les lymphocytes TCD4 hdper et CD8 cytotoxiques. La mise au point d'adjuvants et de molécules mimant un agent pathogène tels les ligands TLRs ou d'autres agents facilitant l'internalisation d'antigènes, est essentielle pour casser la tolérance du système immunitaire contre les cellules cancéreuses afin de générer une réponse effectrice et mémoire contre la tumeur. L'utilisation de longs peptides synthétiques (LSPs) est une approche prometteuse du fait que leur présentation en tant qu'antigénes requiert leur internalisation et leur transformation par les cellules dendrites (DCs, qui sont les mieux à même d'éviter la tolérance immunitaire. Récemment une sous-population de DCs exprimant le récepteur XCR1 a été décrite comme ayant une capacité supérieure dans la cross-présentation d'antigènes, d'où un intérêt à développer des vaccins ciblant les DCs exprimant le XCR1. Durant ma thèse de doctorat, j'ai exploré différentes approches pour optimiser les vaccins avec LSPs. La première partie visait à cibler les XCR1-DCs à l'aide de la chemokine XCL1 spécifique du récepteur XCR1, soit sou s la forme de protéine de fusion XCL1-OVA LSP-Fc, soit associée à des nanoparticules. La deuxième partie a consisté à tester l'association des LSPs avec I adjuvant QS21 dérivant d'une saponine dans le but d'optimiser l'internalisation cytosolique des longs peptides. Les protéines de fusion XCLl-OVA-Fc développées dans la première partie de mon travail, ont démontré leur capacité de liaison spécifique sur les XCRl-DCs associée à leur capacité de chemo-attractio. Lorsque inclues dans une mmunisation de souris porteuse de tumeurs établies, ces protéines de fusion XCL1-0VA LSP-Fc et XCLl-Fc plus OVA LSP ont induites une forte réponse CDS OVA spécifique permettant la complète régression des tumeurs de modèle EL4- 0VA et un retard de croissance significatif de tumeurs de type B16-0VA. Dans le but d'optimiser le drainage des LSPs vers es noyaux lymphatiques, nous avons également testé les LSPs fixés de manière covalente à des nanoparticules co- injectees ou non avec la chemokine XCL1. Cette formulation a également permis une forte réponse CD8 accompagnée d'un effet thérapeutique significatif, mais l'addition de la chemokine XCL1 n'a pas ajouté d'effet anti-tumeur supplémentaire. Dans la deuxième partie de ma thèse, j'ai comparé l'immunogénicité de l'antigène humain Melan A soit sous la forme d un LSP incluant un épitope CD4 et CD8 ou sous la forme d'un peptide ne contenant que l'épitope CD8 (SSP) Les peptides ont été formulés avec l'adjuvant QS21 et testés dans un modèle de souris transgéniques pour les MHC let II humains, respectivement le HLA-A2 et DR1. Les deux peptides LSP et SSP ont généré une forte réponse CD8 similaire assoc.ee a une capacité cytotoxique équivalente lors du transfert in vivo de cellules cibles présentant le peptide SSP' Cependant les souris immunisées avec le Melan A LSP présentaient un pourcentage plus élevé de CD8 ayant un Phénotype «centra, memory» (CD44h' CD62L+ and CCR7+ CD62L+) que les souris immunisées avec le SSP, même dix mois après I'immunisation. Par ailleurs, la réponse CD8 au Melan A LSP était strictement dépendante des lymphocytes CD4, contrairement à l'immunisation par le Melan A SSP qui n'était pas affectée. Dans l'ensemble ces résultats suggèrent que la chemokine XCL1 et l'adjuvant QS21 améliorent la réponse CD8 à un long peptide synthétique, favorisant ainsi le développement d'une réponse anti-tumeur mémoire durable. Ces observations pourraient être utiles au développement de nouveau vaccins thérapeutiques contre les tumeurs.

Intrinsic ROS-production capacity of nanoparticles

Engineered nanomaterials (ENMs) exhibit special physicochemical properties and thus are finding their way into an increasing number of industries, enabling products with improved properties. Their increased use brings a greater likelihood of exposure to the nanoparticles (NPs) that could be released during the life cycle of nano-abled products. The field of nanotoxicology has emerged as a consequence of the development of these novel materials, and it has gained ever more attention due to the urgent need to gather information on exposure to them and to understand the potential hazards they engender. However, current studies on nanotoxicity tend to focus on pristine ENMs, and they use these toxicity results to generalize risk assessments on human exposure to NPs. ENMs released into the environment can interact with their surroundings, change characteristics and exhibit toxicity effects distinct from those of pristine ENMs. Furthermore, NPs' large surface areas provide extra-large potential interfaces, thus promoting more significant interactions between NPs and other co-existing species. In such processes, other species can attach to a NP's surface and modify its surface functionality, in addition to the toxicity in normally exhibits. One particular occupational health scenario involves NPs and low-volatile organic compounds (LVOC), a common type of pollutant existing around many potential sources of NPs. LVOC can coat a NP's surface and then dominate its toxicity. One important mechanism in nanotoxicology is the creation of reactive oxygen species (ROS) on a NP's surface; LVOC can modify the production of these ROS. In summary, nanotoxicity research should not be limited to the toxicity of pristine NPs, nor use their toxicity to evaluate the health effects of exposure to environmental NPs. Instead, the interactions which NPs have with other environmental species should also be considered and researched. The potential health effects of exposure to NPs should be derived from these real world NPs with characteristics modified by the environment and their distinct toxicity. Failure to suitably address toxicity results could lead to an inappropriate treatment of nano- release, affect the environment and public health and put a blemish on the development of sustainable nanotechnologies as a whole. The main objective of this thesis is to demonstrate a process for coating NP surfaces with LVOC using a well-controlled laboratory design and, with regard to these NPs' capacity to generate ROS, explore the consequences of changing particle toxicity. The dynamic coating system developed yielded stable and replicable coating performance, simulating an important realistic scenario. Clear changes in the size distribution of airborne NPs were observed using a scanning mobility particle sizer, were confirmed using both liquid nanotracking analyses and transmission electron microscopy (TEM) imaging, and were verified thanks to the LVOC coating. Coating thicknesses corresponded to the amount of coating material used and were controlled using the parameters of the LVOC generator. The capacity of pristine silver NPs (Ag NPs) to generate ROS was reduced when they were given a passive coating of inert paraffin: this coating blocked the reactive zones on the particle surfaces. In contrast, a coating of active reduced-anthraquinone contributed to redox reactions and generated ROS itself, despite the fact that ROS generation due to oxidation by Ag NPs themselves was quenched. Further objectives of this thesis included development of ROS methodology and the analysis of ROS case studies. Since the capacity of NPs to create ROS is an important effect in nanotoxicity, we attempted to refine and standardize the use of 2'7-dichlorodihydrofluorescin (DCFH) as a chemical tailored for the characterization of NPs' capacity for ROS generation. Previous studies had reported a wide variety of results, which were due to a number of insufficiently well controlled factors. We therefore cross-compared chemicals and concentrations, explored ways of dispersing NP samples in liquid solutions, identified sources of contradictions in the literature and investigated ways of reducing artificial results. The most robust results were obtained by sonicating an optimal sample of NPs in a DCFH-HRP solution made of 5,M DCFH and 0.5 unit/ml horseradish peroxidase (HRP). Our findings explained how the major reasons for previously conflicting results were the different experimental approaches used and the potential artifacts appearing when using high sample concentrations. Applying our advanced DCFH protocol with other physicochemical characterizations and biological analyses, we conducted several case studies, characterizing aerosols and NP samples. Exposure to aged brake wear dust engenders a risk of potential deleterious health effects in occupational scenarios. We performed microscopy and elemental analyses, as well as ROS measurements, with acellular and cellular DCFH assays. TEM images revealed samples to be heterogeneous mixtures with few particles in the nano-scale. Metallic and non-metallic elements were identified, primarily iron, carbon and oxygen. Moderate amounts of ROS were detected in the cell-free fluorescent tests; however, exposed cells were not dramatically activated. In addition to their highly aged state due to oxidation, the reason aged brake wear samples caused less oxidative stress than fresh brake wear samples may be because of their larger size and thus smaller relative reactive surface area. Other case studies involving welding fumes and differently charged NPs confirmed the performance of our DCFH assay and found ROS generation linked to varying characteristics, especially the surface functionality of the samples. Les nanomatériaux manufacturés (ENM) présentent des propriétés physico-chimiques particulières et ont donc trouvés des applications dans un nombre croissant de secteurs, permettant de réaliser des produits ayant des propriétés améliorées. Leur utilisation accrue engendre un plus grand risque pour les êtres humains d'être exposés à des nanoparticules (NP) qui sont libérées au long de leur cycle de vie. En conséquence, la nanotoxicologie a émergé et gagné de plus en plus d'attention dû à la nécessité de recueillir les renseignements nécessaires sur l'exposition et les risques associés à ces nouveaux matériaux. Cependant, les études actuelles sur la nanotoxicité ont tendance à se concentrer sur les ENM et utiliser ces résultats toxicologiques pour généraliser l'évaluation des risques sur l'exposition humaine aux NP. Les ENM libérés dans l'environnement peuvent interagir avec l'environnement, changeant leurs caractéristiques, et montrer des effets de toxicité distincts par rapport aux ENM originaux. Par ailleurs, la grande surface des NP fournit une grande interface avec l'extérieur, favorisant les interactions entre les NP et les autres espèces présentes. Dans ce processus, d'autres espèces peuvent s'attacher à la surface des NP et modifier leur fonctionnalité de surface ainsi que leur toxicité. Un scénario d'exposition professionnel particulier implique à la fois des NP et des composés organiques peu volatils (LVOC), un type commun de polluant associé à de nombreuses sources de NP. Les LVOC peuvent se déposer sur la surface des NP et donc dominer la toxicité globale de la particule. Un mécanisme important en nanotoxicologie est la création d'espèces réactives d'oxygène (ROS) sur la surface des particules, et les LVOC peuvent modifier cette production de ROS. En résumé, la recherche en nanotoxicité ne devrait pas être limitée à la toxicité des ENM originaux, ni utiliser leur toxicité pour évaluer les effets sur la santé de l'exposition aux NP de l'environnement; mais les interactions que les NP ont avec d'autres espèces environnementales doivent être envisagées et étudiées. Les effets possibles sur la santé de l'exposition aux NP devraient être dérivés de ces NP aux caractéristiques modifiées et à la toxicité distincte. L'utilisation de résultats de toxicité inappropriés peut conduire à une mauvaise prise en charge de l'exposition aux NP, de détériorer l'environnement et la santé publique et d'entraver le développement durable des industries de la nanotechnologie dans leur ensemble. L'objectif principal de cette thèse est de démontrer le processus de déposition des LVOC sur la surface des NP en utilisant un environnement de laboratoire bien contrôlé et d'explorer les conséquences du changement de toxicité des particules sur leur capacité à générer des ROS. Le système de déposition dynamique développé a abouti à des performances de revêtement stables et reproductibles, en simulant des scénarios réalistes importants. Des changements clairs dans la distribution de taille des NP en suspension ont été observés par spectrométrie de mobilité électrique des particules, confirmé à la fois par la méthode dite liquid nanotracking analysis et par microscopie électronique à transmission (MET), et a été vérifié comme provenant du revêtement par LVOC. La correspondance entre l'épaisseur de revêtement et la quantité de matériau de revêtement disponible a été démontré et a pu être contrôlé par les paramètres du générateur de LVOC. La génération de ROS dû aux NP d'argent (Ag NP) a été diminuée par un revêtement passif de paraffine inerte bloquant les zones réactives à la surface des particules. Au contraire, le revêtement actif d'anthraquinone réduit a contribué aux réactions redox et a généré des ROS, même lorsque la production de ROS par oxydation des Ag NP avec l'oxygène a été désactivé. Les objectifs associés comprennent le développement de la méthodologie et des études de cas spécifique aux ROS. Etant donné que la capacité des NP à générer des ROS contribue grandement à la nanotoxicité, nous avons tenté de définir un standard pour l'utilisation de 27- dichlorodihydrofluorescine (DCFH) adapté pour caractériser la génération de ROS par les NP. Des etudes antérieures ont rapporté une grande variété de résultats différents, ce qui était dû à un contrôle insuffisant des plusieurs facteurs. Nous avons donc comparé les produits chimiques et les concentrations utilisés, exploré les moyens de dispersion des échantillons HP en solution liquide, investigué les sources de conflits identifiées dans les littératures et étudié les moyens de réduire les résultats artificiels. De très bon résultats ont été obtenus par sonication d'une quantité optimale d'échantillons de NP en solution dans du DCFH-HRP, fait de 5 nM de DCFH et de 0,5 unité/ml de Peroxydase de raifort (HRP). Notre étude a démontré que les principales raisons causant les conflits entre les études précédemment conduites dans la littérature étaient dues aux différentes approches expérimentales et à des artefacts potentiels dus à des concentrations élevées de NP dans les échantillons. Utilisant notre protocole DCFH avancé avec d'autres caractérisations physico-chimiques et analyses biologiques, nous avons mené plusieurs études de cas, caractérisant les échantillons d'aérosols et les NP. La vielle poussière de frein en particulier présente un risque élevé d'exposition dans les scénarios professionnels, avec des effets potentiels néfastes sur la santé. Nous avons effectué des analyses d'éléments et de microscopie ainsi que la mesure de ROS avec DCFH cellulaire et acellulaire. Les résultats de MET ont révélé que les échantillons se présentent sous la forme de mélanges de particules hétérogènes, desquels une faible proportion se trouve dans l'échelle nano. Des éléments métalliques et non métalliques ont été identifiés, principalement du fer, du carbone et de l'oxygène. Une quantité modérée de ROS a été détectée dans le test fluorescent acellulaire; cependant les cellules exposées n'ont pas été très fortement activées. La raison pour laquelle les échantillons de vielle poussière de frein causent un stress oxydatif inférieur par rapport à la poussière de frein nouvelle peut-être à cause de leur plus grande taille engendrant une surface réactive proportionnellement plus petite, ainsi que leur état d'oxydation avancé diminuant la réactivité. D'autres études de cas sur les fumées de soudage et sur des NP différemment chargées ont confirmé la performance de notre test DCFH et ont trouvé que la génération de ROS est liée à certaines caractéristiques, notamment la fonctionnalité de surface des échantillons.

Interplay between surface anisotropy and dipolar interactions in an assembly of nanomagnets

We study the interplay between the effects of surface anisotropy and dipolar interactions in monodisperse assemblies of nanomagnets with oriented anisotropy. We derive asymptotic formulas for the assembly magnetization, taking into account temperature, applied field, core and surface anisotropy, and dipolar interparticle interactions. We find that the interplay between surface anisotropy and dipolar interactions is well described by the analytical expression of the assembly magnetization derived here: the overall sign of the product of the two parameters governing the surface and the dipolar contributions determines whether intrinsic and collective terms compete or have synergistic effects on the magnetization. This is illustrated by the magnetization curves of γ-Fe2O3 nanoparticle assemblies in the low concentration limit.

Behind and beyond of the cyborgs and robots ideas and realities: some techno-scientific and philosophical hints

We are progressively immersed in technology to such extend that in our everyday life we are and we do what technology allows us to be and to do. In this process, cyborgs and robots constitute elements that we analyze from a number of techno scientific and philosophical approaches. Additionally, we propose that a new concept: GEH (Genetic Engineered Human) as a new potential social imaginary element, which would be the human being improved by the broad-sense genetics engineer (that is, changing many genes by genetics engineer, modifications in the genome, cloning, and so on). If our aspirations as humans pass through technology and in particular for cyborgs, robots and GEH, the bidirectional links between these theoretical or real entities and our personal identities will be the more and more substantial in our society.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

The systemic control of circadian gene expression.

The mammalian circadian timing system consists of a central pacemaker in the brain's suprachiasmatic nucleus (SCN) and subsidiary oscillators in nearly all body cells. The SCN clock, which is adjusted to geophysical time by the photoperiod, synchronizes peripheral clocks through a wide variety of systemic cues. The latter include signals depending on feeding cycles, glucocorticoid hormones, rhythmic blood-borne signals eliciting daily changes in actin dynamics and serum response factor (SRF) activity, and sensors of body temperature rhythms, such as heat shock transcription factors and the cold-inducible RNA-binding protein CIRP. To study these systemic signalling pathways, we designed and engineered a novel, highly photosensitive apparatus, dubbed RT-Biolumicorder. This device enables us to record circadian luciferase reporter gene expression in the liver and other organs of freely moving mice over months in real time. Owing to the multitude of systemic signalling pathway involved in the phase resetting of peripheral clocks the disruption of any particular one has only minor effects on the steady state phase of circadian gene expression in organs such as the liver. Nonetheless, the implication of specific pathways in the synchronization of clock gene expression can readily be assessed by monitoring the phase-shifting kinetics using the RT-Biolumicorder.

Estudi de noves estratègies encapsuladores d’antimalàrics i distribució de nanovectors polimèrics en Anopheles atroparvus

En aquest treball s’ha estudiat el comportament de compostos antimalàrics com els fàrmacs i els polímers en diferents situacions. Una de les barreres que ha estat identificada com a principal obstacle per a una millora de l’eficàcia dels compostos antimalàrics, és la limitació en la quantitat de fàrmac que pot ser encapsulada dins un liposoma, i que depèn de la seva solubilitat en medi aquós. Amb la inspiració de la descripció d’un nou tipus de nanocàpsules amb aplicacions oncològiques capaces d’encapsular grans quantitats de fàrmacs (protocells, Ashley et al., 2011). Els constructes formats per liposomes amb un nucli d’òxid de silici altament porós capaç de contenir el fàrmac, s’anomenen “protocells”, que en comparació als liposomes, tenen una major selectivitat i estabilitat, i permeten alliberar altes concentracions de droga directament al citosol de les cèl·lules cancerígenes. Aquest estudi es basa en la fabricació d’aquests nous nanovectors que continguin fàrmacs antimalàrics i té com a objectiu futur dirigir-los a eritròcits infectats per malària (pRBCs). Una altra part del treball és l’estudi de la distribució del polímer ISA-FITC en Anopheles atroparvus. Sabent que els polímers han estat utilitzats com a transportadors antimalàrics, es va pensar en l’opció d’eliminar el paràsit a dins del mateix mosquit, com una alternativa a tots el estudis realitzats fins ara centrats en les etapes d’infecció de l’hoste. Per aquest motiu es va idear l’experiment pensant en aquest polímer amb la intenció final de veure la seva localització en un mosquit Anopheles lliure del paràsit. OBJECTIUS: Determinació de la capacitat encapsuladora de tres tipus de nanopartícules, fabricades amb el mateix material però amb característiques de mida i càrrega diferents, incubant-les amb cinc fàrmacs antimalàrics. El blau de metilè, la primaquina, la cloroquina, la quinina i la curcumina, cadascun d’ells amb característiques de pH, solubilitat i estructura diferents. Alguns d’ells són fàrmacs que no s’han emprat en altres estudis degut a la seva toxicitat o elevada inespecificitat (la qual es pretén reduir un cop encapsulats en protocells). Construcció de “protocells” un cop determinada la millor nanopartícula encapsuladora i fàrmac candidat i determinació de la concentració de fàrmac que podien contenir, i el ritme d’alliberament d’aquest en PBS (simulant les condicions fisiològiques dels pRBCs). Estudi de la localització del polímer antimalàric ISA-FITC en l’anatomia del mosquit Anopheles Atroparvus. PROCEDIMENTS: Mètodes espectrofotomètrics Microscopia Cryo-electrònica de transmissió Microscopia confocal de fluorescència

Apical closure device for transapical valve procedures?.

OBJECTIVES: Transapical transcatheter valve procedures are performed through a left minithoracotomy and require apical sutures to seal the apical access site. The use of large-calibre devices compromises any attempt to fully perform the procedure with a thoracoscopic approach or percutaneously. We report our preliminary experience in animals with a new sutureless self-expandable apical occluder, engineered to perform transapical access site closure in a minimally invasive setting with large-size introducer sheaths. METHODS: The apical occluder with extendable waist was implanted in six young pigs during an acute animal study. Under general anaesthesia, animals (mean weight: 62 ± 8 kg) received full heparinization (heparin: 100 UI/kg; activated clotting time above 250 s). Through a median sternotomy, a 21-Fr Certitude? introducer sheath (outer diameter: 25 Fr) was placed over the wire into the cardiac apex. The delivery catheter carrying the constrained apical plug was inserted into the sheath and deployed under fluoroscopic control, whereas the Certitude? was retrieved. After protamine infusion, we observed and recorded the 1-h bleeding with standard haemodynamic parameters. Animals were sacrificed, and hearts analysed. RESULTS: Six apical closure devices were successfully introduced and deployed in six pig hearts through large-size apical sheaths at first attempt. In all animals, the plugs guaranteed immediate apical sealing and traces of blood were collected in the pericardium during the 1-h observational period (mean of 16 ± 3.4 ml of blood loss per animal). Haemodynamic parameters remained stable during the entire study period and no plug dislodgement was detected with normal systemic blood pressure (mean arterial mean blood pressure: 65 ± 7 mmHg). Post-mortem analysis confirmed the full deployment and good fixation of all plugs, without macroscopic damages to the surrounding myocardium. CONCLUSIONS: This sutureless self-expandable apical occluder is a simple device capable of sealing large-size apical access sites (20-35 Fr) in an acute animal study. This approach is a step further towards less invasive transapical valve procedures in the clinical setting, and further animal tests will be performed to confirm the long-term efficacy and safety of this device.

Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production.

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.

Structure-Based, Rational Design of T Cell Receptors.

Adoptive cell transfer using engineered T cells is emerging as a promising treatment for metastatic melanoma. Such an approach allows one to introduce T cell receptor (TCR) modifications that, while maintaining the specificity for the targeted antigen, can enhance the binding and kinetic parameters for the interaction with peptides (p) bound to major histocompatibility complexes (MHC). Using the well-characterized 2C TCR/SIYR/H-2K(b) structure as a model system, we demonstrated that a binding free energy decomposition based on the MM-GBSA approach provides a detailed and reliable description of the TCR/pMHC interactions at the structural and thermodynamic levels. Starting from this result, we developed a new structure-based approach, to rationally design new TCR sequences, and applied it to the BC1 TCR targeting the HLA-A2 restricted NY-ESO-1157-165 cancer-testis epitope. Fifty-four percent of the designed sequence replacements exhibited improved pMHC binding as compared to the native TCR, with up to 150-fold increase in affinity, while preserving specificity. Genetically engineered CD8(+) T cells expressing these modified TCRs showed an improved functional activity compared to those expressing BC1 TCR. We measured maximum levels of activities for TCRs within the upper limit of natural affinity, K D = ∼1 - 5 μM. Beyond the affinity threshold at K D < 1 μM we observed an attenuation in cellular function, in line with the "half-life" model of T cell activation. Our computer-aided protein-engineering approach requires the 3D-structure of the TCR-pMHC complex of interest, which can be obtained from X-ray crystallography. We have also developed a homology modeling-based approach, TCRep 3D, to obtain accurate structural models of any TCR-pMHC complexes when experimental data is not available. Since the accuracy of the models depends on the prediction of the TCR orientation over pMHC, we have complemented the approach with a simplified rigid method to predict this orientation and successfully assessed it using all non-redundant TCR-pMHC crystal structures available. These methods potentially extend the use of our TCR engineering method to entire TCR repertoires for which no X-ray structure is available. We have also performed a steered molecular dynamics study of the unbinding of the TCR-pMHC complex to get a better understanding of how TCRs interact with pMHCs. This entire rational TCR design pipeline is now being used to produce rationally optimized TCRs for adoptive cell therapies of stage IV melanoma.

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

Extracellular Subunit Interactions Control Transitions between Functional States of Acid-sensing Ion Channel 1a.

Acid-sensing ion channels (ASICs) are neuronal, voltage-independent Na(+) channels that are transiently activated by extracellular acidification. They are involved in pain sensation, the expression of fear, and in neurodegeneration after ischemic stroke. Our study investigates the role of extracellular subunit interactions in ASIC1a function. We identified two regions involved in critical intersubunit interactions. First, formation of an engineered disulfide bond between the palm and thumb domains leads to partial channel closure. Second, linking Glu-235 of a finger loop to either one of two different residues of the knuckle of a neighboring subunit opens the channel at physiological pH or disrupts its activity. This suggests that one finger-knuckle disulfide bond (E235C/K393C) sets the channel in an open state, whereas the other (E235C/Y389C) switches the channel to a non-conducting state. Voltage-clamp fluorometry experiments indicate that both the finger loop and the knuckle move away from the β-ball residue Trp-233 during acidification and subsequent desensitization. Together, these observations reveal that ASIC1a opening is accompanied by a distance increase between adjacent thumb and palm domains as well as a movement of Glu-235 relative to the knuckle helix. Our study identifies subunit interactions in the extracellular loop and shows that dynamic changes of these interactions are critical for normal ASIC function.