Atividade antiofídica do decocto das folhas de jatropha gossypiifolia l. frente o veneno de bothrops jararaca

Antiophidic activity from decoct of Jatropha gossypiifolia L. leaves against Bothrops jararaca venom. Snakebites are a serious worldwide public health problem. In Latin America, about 90 % of accidents are attributed to snakes from Bothrops genus. Currently, the main available treatment is the antivenom serum therapy, which has some disadvantages such as inability to neutralize local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies to treat snakebites is relevant. Jatropha gossypiifolia L., a medicinal plant popularly known in Brazil as “pinhão-roxo”, is very used in folk medicine as antiophidic. So, the aim of this study is to evaluate the antiophidic properties of this species against enzymatic and biological activities from Bothrops jararaca snake venom. The aqueous leaf extract of J. gossypiifolia was prepared by decoction. The inhibition studies were performed in vitro, by pre-incubation of a fixed amount of venom with different amounts of extract from J. gossypiifolia for 60 min at 37 °C, and in vivo, through oral or intraperitoneal treatment of animals, in different doses, 60 min before venom injection. The proteolytic activity upon azocasein was efficiently inhibited, indicating inhibitory action upon metalloproteinases (SVMPs) and/or serine proteases (SVSPs). The extract inhibited the fibrinogenolytic activity, which was also confirmed by zymography, where it was possible to observe that the extract preferentially inhibits fibrinogenolytic enzymes of 26 and 28 kDa. The coagulant activity upon fibrinogen and plasma were significantly inhibited, suggesting an inhibitory action upon thrombin-like enzymes (SVTLEs), as well as upon clotting factor activators toxins. The extract prolonged the activated partial thromboplastin time (aPTT), suggesting an inhibitory action toward not only to SVTLEs, but also against endogenous thrombin. The defibrinogenating activity in vivo was efficiently inhibited by the extract on oral route, confirming the previous results. The local hemorrhagic activity was also significantly inhibited by oral route, indicating an inhibitory action upon SVMPs. The phospholipase activity in vitro was not inhibited. Nevertheless, the edematogenic and myotoxic activities were efficiently inhibited, by oral and intraperitoneal route, which may indicate an inhibitory effect of the extract upon Lys49 phospholipase (PLA2) and/ or SVMPs, or also an anti-inflammatory action against endogenous chemical mediators. Regarding the possible action mechanism, was observed that the extract did not presented proteolytic activity, however, presented protein precipitating action. In addition, the extract showed significant antioxidant activity in different models, which could justify, at least partially, the antiophidic activity presented. The metal chelating action presented by extract could be correlated with SVMPs inhibition, once these enzymes are metal-dependent. The phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins, from which the flavonoids could be pointed as major compounds, based on chromatographic profile obtained by thin layer chromatography (TLC). In conclusion, the results demonstrate that the J. gossypiifolia leaves decoct present potential antiophidic activity, including action upon snakebite local effects, suggesting that this species may be used as a new source of bioactive molecules against bothropic venom.

Visualizing Rare Watson-Crick-Like Tautomeric and Anionic Mismatches in DNA and RNA

The central dogma of molecular biology relies on the correct Watson-Crick (WC) geometry of canonical deoxyribonucleic acid (DNA) dG•dC and dA•dT base pairs to replicate and transcribe genetic information with speed and an astonishing level of fidelity. In addition, the Watson-Crick geometry of canonical ribonucleic acid (RNA) rG•rC and rA•rU base pairs is highly conserved to ensure that proteins are translated with high fidelity. However, numerous other potential nucleobase tautomeric and ionic configurations are possible that can give rise to entirely new pairing modes between the nucleotide bases. Very early on, James Watson and Francis Crick recognized their importance and in 1953 postulated that if bases adopted one of their less energetically disfavored tautomeric forms (and later ionic forms) during replication it could lead to the formation of a mismatch with a Watson-Crick-like geometry and could give rise to “natural mutations.”

Since this time numerous studies have provided evidence in support of this hypothesis and have expanded upon it; computational studies have addressed the energetic feasibilities of different nucleobases’ tautomeric and ionic forms in siico; crystallographic studies have trapped different mismatches with WC-like geometries in polymerase or ribosome active sites. However, no direct evidence has been given for (i) the direct existence of these WC-like mismatches in canonical DNA duplex, RNA duplexes, or non-coding RNAs; (ii) which, if any, tautomeric or ionic form stabilizes the WC-like geometry. This thesis utilizes nuclear magnetic resonance (NMR) spectroscopy and rotating frame relaxation dispersion (R1ρ RD) in combination with density functional theory (DFT), biochemical assays, and targeted chemical perturbations to show that (i) dG•dT mismatches in DNA duplexes, as well as rG•rU mismatches RNA duplexes and non-coding RNAs, transiently adopt a WC-like geometry that is stabilized by (ii) an interconnected network of rapidly interconverting rare tautomers and anionic bases. These results support Watson and Crick’s tautomer hypothesis, but additionally support subsequent hypotheses invoking anionic mismatches and ultimately tie them together. This dissertation shows that a common mismatch can adopt a Watson-Crick-like geometry globally, in both DNA and RNA, and whose geometry is stabilized by a kinetically linked network of rare tautomeric and anionic bases. The studies herein also provide compelling evidence for their involvement in spontaneous replication and translation errors.

Insulin-like growth factor receptor activity in cancer biology & therapy responses

The Insulin-like Growth Factor 1 Receptor (IGF-1R) has an essential function in normal cell growth and in cancer progression. However, anti-IGF-1R therapies have mostly been withdrawn from clinical trials owing to a lack of efficacy and predictive biomarkers. IGF-1R activity and signalling in cancer cells is regulated by its C-terminal tail, and in particular, by a motif that encompasses tyrosines 1250 and 1251 flanked by serines 1248 and 1252 (1248- SFYYS-1252). Mutation of Y1250/1251 greatly reduces IGF-1-promoted cell migration, interaction with the scaffolding protein RACK1 in the context Integrin signalling, and IGF- 1R kinase activity. Here we investigated the phosphorylation of the SFYYS motif and characterise the conditions under which this motif may be phosphorylated under. As phosphorylated residues, the SFYYS motif may also serve to recruit interacting proteins to the IGF-1R. To this end we identified a novel IGF-1R interacting partner which requires phosphorylated residues in the SFYYS motif to interact with the IGF-1R. This interaction was found to be IGF-1-dependent, and required the scaffold protein RACK1. The interaction of this binding protein with the IGF-1R likely functions to promote maximal phosphorylation of Shc and ERK in IGF-1-stimulated cell migration, and may be important for IGF-1 signalling in cancer cells. Lastly, we have investigated possible kinases that may confer resistance or sensitivity to the IGF-1R kinase inhibitor BMS-754807. In this screen we identified ATR as a mediator of resistance and showed that suppression or chemical inhibition of ATR synergised with BMS-754807 to reduce colony formation. This work has contributes to our understanding of IGF-1R kinase regulation and signalling and suggests that administration of anti-IGF-1R drugs with ATR inhibitors may have therapeutic benefit.

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets

Background: Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.

Methods: Washed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.

Results: Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) PKA-mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.

Conclusions: We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Defensin-Like ZmES4 Mediates Pollen Tube Burst in Maize via Opening of the Potassium Channel KZM1

In contrast to animals and lower plant species, sperm cells of flowering plants are non-motile and are transported to the female gametes via the pollen tube, i.e. the male gametophyte. Upon arrival at the female gametophyte two sperm cells are discharged into the receptive synergid cell to execute double fertilization. The first players involved in inter-gametophyte signaling to attract pollen tubes and to arrest their growth have been recently identified. In contrast the physiological mechanisms leading to pollen tube burst and thus sperm discharge remained elusive. Here, we describe the role of polymorphic defensin-like cysteine-rich proteins ZmES1-4 (Zea mays embryo sac) from maize, leading to pollen tube growth arrest, burst, and explosive sperm release. ZmES1-4 genes are exclusively expressed in the cells of the female gametophyte. ZmES4-GFP fusion proteins accumulate in vesicles at the secretory zone of mature synergid cells and are released during the fertilization process. Using RNAi knock-down and synthetic ZmES4 proteins, we found that ZmES4 induces pollen tube burst in a species-preferential manner. Pollen tube plasma membrane depolarization, which occurs immediately after ZmES4 application, as well as channel blocker experiments point to a role of K(+)-influx in the pollen tube rupture mechanism. Finally, we discovered the intrinsic rectifying K(+) channel KZM1 as a direct target of ZmES4. Following ZmES4 application, KZM1 opens at physiological membrane potentials and closes after wash-out. In conclusion, we suggest that vesicles containing ZmES4 are released from the synergid cells upon male-female gametophyte signaling. Subsequent interaction between ZmES4 and KZM1 results in channel opening and K(+) influx. We further suggest that K(+) influx leads to water uptake and culminates in osmotic tube burst. The species-preferential activity of polymorphic ZmES4 indicates that the mechanism described represents a pre-zygotic hybridization barrier and may be a component of reproductive isolation in plants.

A study of fish ice cream formulation by use of silver carp proteins instead of milk proteins

Fish is a valuable nutritional source witch use of it in daily meal has a beneficial role on nutritional needs supply and also causes mental and physical health especially in people who have protein and food deficiencies. Unfortunately, per capita consumption of sea foods in Iran is 5.5Kg witch is very lower than world standards. So, study on fish ice cream formulation, by use of fish protein concentrate (FPC) instead of milk protein, had done to make variation in sea foods products and also increase per capita consumption of these kinds of foods. FPC has very high protein concentration and a lot of necessary Also it's protein is very digestible amino acids like lysine and methionine with highly biological value and it's PER in compare with casein PER is high. At first fish protein concentrate type A produced from silver carp in three steps by the extraction with isopropyl alcohol solvent and heat. Microbiological and physicochemical specifications of produced FPC by rules of FDA and FAO were accepted. Finally according to panel test results, substitution of 30 percent of milk with FPC is acceptable. Also microbiological and physicochemical specifications of product were tested and results in compare with national standards of Iran were accepted.

CD81 interacting proteins

Fertilization is a multistep and complex process culminating in the merge of gamete membranes, cytoplasmic unity and fusion of genome. CD81 is a tetraspanin protein that participates in sperm-oocyte interaction, being present at the oocyte surface. CD81 has also been implicated in other biological processes, however its specific function and molecular mechanisms of action remain to be elucidated. The interaction between CD81 and its binding partner proteins may underlie the CD81 involvement in a variety of cellular processes and modulate CD81/interactors specific functions. Interestingly, in a Yeast two Hybrid system previously performed in our lab, CD81 has emerged as a putative interactor of the Amyloid Precursor Protein (APP). In the work here described, bioinformatics analyses of CD81 interacting proteins were performed and the retrieved information used to construct a protein-protein interaction network, as well as to perform Gene Ontology enrichment analyses. CD81 expression was further evaluated in CHO, GC-1 and SH-SY5Y cell lines, and in human sperm cells. Additionally, its subcellular localization was analyzed in sperm cells and in the neuronal-like SH-SY5Y cell line. Subsequently, coimmunoprecipitation assays were performed in CHO and SH-SY5Y cells to attempt to prove the physical interaction between CD81 and APP. A functional interaction between these two proteins was accessed thought the analyses of the effects of CD81 overexpression on APP levels. A co-localization analysis of CD81 and some interactors proteins retrieved from the bioinformatics analyses, such as APP, AKT1 and cytoskeleton-related proteins, was also performed in sperm cells and in SH-SY5Y cells. The effects of CD81 in cytoskeleton remodeling was evaluated in SH-SY5Y cells through monitoring the effects of CD81 overexpression in actin and tubulin levels, and analyzing the colocalization between overexpressed CD81 and F-actin. Our results showed that CD81 is expressed in all cell lines tested, and also provided the first evidence of the presence of CD81 in human sperm cells. CD81 immunoreactivity was predominantly detected in the sperm head, including the acrosome membrane, and in the midpiece, where it co-localized with APP, as well as in the post-acrosomal region. Furthermore, CD81 co-localizes with APP in the plasma membrane and in cellular projections in SH-SY5Y cells, where CD81 overexpression has an influence on APP levels, also visible in CHO cells. The analysis of CD81 interacting proteins such as AKT1 and cytoskeletonrelated proteins showed that CD81 is involved in a variety of pathways that may underlie cytoskeleton remodeling events, related to processes such as sperm motility, cell migration and neuritogenesis. These results deepen our understanding on the functions of CD81 and some of its interactors in sperm and neuronal cells.

KINESIN MOTOR PROTEINS ARE ESSENTIAL FOR MALE GAMETOPHYTE DEVELOPMENT IN MARSILEA VESTITA

The male gametophyte of the semi-aquatic fern, Marsilea vestita, produces multiciliated spermatozoids in a rapid developmental sequence that is controlled post-transcriptionally when dry microspores are placed in water. Development can be divided into two phases, mitosis and differentiation. During the mitotic phase, a series of nine successive division cycles produce 7 sterile cells and 32 spermatids in 4.5-5 hours. During the next 5-6 hours, each spermatid differentiates into a corkscrew-shaped motile spermatozoid with ~140 cilia. This document focuses on the role of motor proteins in the regulation of male gametophyte development and during ciliogenesis. In order to study the mechanisms that regulate spermatogenesis, RNAseq was used to generate a reference transcriptome that allowed us to assess the abundance of transcripts at different stages of development. Over 120 kinesin-like sequences were identified in the transcriptome that represent 56 unique kinesin transcripts. Members of the kinesin-2, -4, -5, -7, -8, -9, -12, -13, and -14 families, in addition to several plant specific and ‘orphan’ kinesins are present. Most (91%) of these kinesin transcripts change in abundance throughout gametophyte development, with 52% of kinesin mRNAs enriched during the mitotic phase and 39% enriched during differentiation. Functional analyses show that the temporal regulation of kinesin transcripts during gametogenesis directly correlates with kinesin protein function. Specifically, Marsilea makes one kinesin-2 (MvKinesin-2) and two kinesin-9 (MvKinesin-9A and MvKinesin-9B) transcripts, which are present during spermatid differentiation and ciliogenesis. Silencing experiments showed that MvKinesin-2 and MvKinesin-9A are required for ciliogenesis and motility in the Marsilea male gametophyte; however, these kinesins display atypical roles during these processes. In contrast, spermatozoids produced after the silencing of MvKinesin-9B exhibit normal morphology. MvKinesin-2 is necessary for cytokinesis as well as for regulating ciliary length and MvKinesin-9A is needed for the correct orientation of basal bodies, events not typically associated with these proteins. In addition, Marsilea makes motile, ciliated gametophytes without the help of IFT dynein, outer arm dynein, or the BBsome. These results are the first to investigate the kinesin-linked mechanisms that regulate ciliogenesis in a land plant.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins

International audience

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Purification and characterization of the biological effects of phospholipase A(2) from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA(2) isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the Sao Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA(2) proteins (named BcPLA(2)1, BCPLA(2)2 and BcPLA(2)3) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA(2)1 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA(2)1 showed high amino acid sequence identity with PLA(2) group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA(2) and 1POC_A). In addition, BcPLA(2)1 also showed significant overall homology to bee PLA(2). The enzymatic activity induced by native BCPLA(2)1 (20 mu g/well) was reduced by chemical treatment with p-bromophenacyl bromide (p-BPB) and with morin. BcPLA(2)1 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA(2) from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA(2)1, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA(2), a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration. (C) 2009 Elsevier Ltd. All rights reserved.

Positive biocompatibility of nanocrystalline glass-like carbon thin films

The interest in carbon nanomaterials with high transparency and electrical conductivity has grown within the last decade in view of a wide variety of applications, including biocompatible sensors, diagnostic devices and bioelectronic implants. The aim of this work is to test the biocompatibility of particular nanometer-thin nanocrystalline glass-like carbon films (NGLC), a disordered structure of graphene flakes joined by carbon matrix (Romero et al., 2016). We used a cell line (SN4741) from substantia nigra dopaminergic cells derived from transgenic mouse embryo cells (Son et al., 1999). Some cells were cultured on top of NGLC films (5, 20 and 80 nm) and other with NGLC nanoflakes (approx. 5-10 mm2) in increasing concentrations: 1, 5, 10, 20 and 50 μg/ml, during 24 h, 3 days and 7 days. Cells growing in normal conditions were defined under culture with DMEM supplemented with 10% FCS, Glucose (0,6%), penicillin-streptomycin (50U/ml) and L-glutamine (2mM) at 5%CO2 humidified atmosphere. Nanoflakes were resuspended in DMEM at the stock concentration (2 g/l). The experiments were conducted in 96 well plates (Corning) using 2500 cells per well. For MTT analysis, the manufacturer recommendations were followed (Roche, MTT kit assay): a positive control with a 10% Triton X-100 treatments (15 minutes) and a negative control without neither Triton X-100 nor NGLC. As apoptosis/necrosis assay we used LIVE/DEAD® Viability/Cytotoxicity Assay Kit (Invitrogen). In a separate experiment, cells were cultured on top of the NGLC films for 7 days. Primary antibodies: anti-synaptophysin (SYP, clone SY38, Chemicon) and goat anti-GIRK2 (G-protein-regulated inward-rectifier potassium channel 2 protein) (Abcom) following protocol for immunofluorescence. WB for proteins detection performed with a polyclonal anti-rabbit proliferating cell nuclear antigen (PCNA). Results demonstrated the biocompatibility with different concentration of NGLC varying the degree of survival from a low concentration (1 mg/ml) in the first 24 h to high concentrations (20-50 g/ml) after 7 days as it is corroborated by the PCNA analysis. Cells cultured on top of the film showed after 7 days axonal-like alignment and edge orientation as well as net-like images. Neuronal functionality was demonstrated to a certain extent through the analysis of coexistence between SYP and GIRK2. In conclusion, this nanomaterial could offer a powerful platform for biomedical applications such as neural tissue engineering

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.

Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep

BACKGROUND Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. METHODOLOGY/PRINCIPAL FINDINGS Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. CONCLUSIONS There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if immunodominance of particular serotypes could interfere with vaccine efficacy.

Generation and screening of natural product-like compounds for antibiotic discovery

Avec l’apparition de plus en plus de souches de bactérie résistante aux antibiotiques, le développement de nouveaux antibiotiques est devenu une important problématique pour les agences de santé. C’est pour cela que la création de nouvelles plateformes pour accélérer la découverte de médicaments est devenu un besoin urgent. Dans les dernières décennies, la recherche était principalement orientée sur la modification de molécules préexistantes, la méta-analyse d’organismes produisant des molécules activent et l’analyse de librairies moléculaires pour trouver des molécules synthétiques activent, ce qui s’est avéré relativement inefficace. Notre but était donc de développer de nouvelles molécules avec des effets thérapeutiques de façon plus efficace à une fraction du prix et du temps comparé à ce qui se fait actuellement. Comme structure de base, nous avons utilisé des métabolites secondaires qui pouvaient altérer le fonctionnement des protéines ou l’interaction entre deux protéines. Pour générer ces molécules, j’ai concentré mes efforts sur les terpènes, une classe de métabolites secondaires qui possède un large éventail d’activités biologiques incluant des activités antibactériennes. Nous avons développé un système de chromosome artificiel de levure (YAC) qui permet à la fois l’assemblage directionnel et combinatoire de gènes qui permet la création de voies de biosynthèse artificielles. Comme preuve de concept, j’ai développé des YACs qui contiennent les gènes pour l’expression des enzymes impliquées dans la biosynthèse de la -carotène et de l’albaflavenone et produit ces molécules avec un haut rendement. Finalement, Des YACs produits à partir de librairies de gènes ont permis de créer une grande diversité de molécules.