Calcium imaging of odor-evoked responses in the Drosophila antennal lobe.

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions. Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP and Cameleon, the bioluminescent calcium indicator GFP-aequorin, or a reporter of synaptic transmission, synapto-pHluorin has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS, LexA/LexAop, Q system) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed. Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Monoclonal auto-antibodies and sera of autoimmune patients react with Plasmodium falciparum and inhibit its in vitro growth

The relationship between autoimmunity and malaria is not well understood. To determine whether autoimmune responses have a protective role during malaria, we studied the pattern of reactivity to plasmodial antigens of sera from 93 patients with 14 different autoimmune diseases (AID) who were not previously exposed to malaria. Sera from patients with 13 different AID reacted against Plasmodium falciparum by indirect fluorescent antibody test with frequencies varying from 33-100%. In addition, sera from 37 AID patients were tested for reactivity against Plasmodium yoelii 17XNL and the asexual blood stage forms of three different P. falciparum strains. In general, the frequency of reactive sera was higher against young trophozoites than schizonts (p < 0.05 for 2 strains), indicating that the antigenic determinants targeted by the tested AID sera might be more highly expressed by the former stage. The ability of monoclonal auto-antibodies (auto-Ab) to inhibit P. falciparum growth in vitro was also tested. Thirteen of the 18 monoclonal auto-Ab tested (72%), but none of the control monoclonal antibodies, inhibited parasite growth, in some cases by greater than 40%. We conclude that autoimmune responses mediated by auto-Ab may present anti-plasmodial activity.

Prevalence and Clinical Outcomes for Patients With ALK-Positive Resected Stage I to III Adenocarcinoma: Results From the European Thoracic Oncology Platform Lungscape Project.

PURPOSE: The prevalence of anaplastic lymphoma kinase (ALK) gene fusion (ALK positivity) in early-stage non-small-cell lung cancer (NSCLC) varies by population examined and detection method used. The Lungscape ALK project was designed to address the prevalence and prognostic impact of ALK positivity in resected lung adenocarcinoma in a primarily European population. METHODS: Analysis of ALK status was performed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) in tissue sections of 1,281 patients with adenocarcinoma in the European Thoracic Oncology Platform Lungscape iBiobank. Positive patients were matched with negative patients in a 1:2 ratio, both for IHC and for FISH testing. Testing was performed in 16 participating centers, using the same protocol after passing external quality assessment. RESULTS: Positive ALK IHC staining was present in 80 patients (prevalence of 6.2%; 95% CI, 4.9% to 7.6%). Of these, 28 patients were ALK FISH positive, corresponding to a lower bound for the prevalence of FISH positivity of 2.2%. FISH specificity was 100%, and FISH sensitivity was 35.0% (95% CI, 24.7% to 46.5%), with a sensitivity value of 81.3% (95% CI, 63.6% to 92.8%) for IHC 2+/3+ patients. The hazard of death for FISH-positive patients was lower than for IHC-negative patients (P = .022). Multivariable models, adjusted for patient, tumor, and treatment characteristics, and matched cohort analysis confirmed that ALK FISH positivity is a predictor for better overall survival (OS). CONCLUSION: In this large cohort of surgically resected lung adenocarcinomas, the prevalence of ALK positivity was 6.2% using IHC and at least 2.2% using FISH. A screening strategy based on IHC or H-score could be envisaged. ALK positivity (by either IHC or FISH) was related to better OS.

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Clinical evaluation of a method for detecting superficial surgical transitional cell carcinoma of the bladder by light-induced fluorescence of protoporphyrin IX following the topical application of 5-aminolevulinic acid: preliminary results.

BACKGROUND AND OBJECTIVE: In bladder cancer, conventional white light endoscopic examination of the bladder does not provide adequate information about the presence of "flat" urothelial lesions such as carcinoma in situ. In the present investigation, we examine a new technique for the photodetection of such lesions by the imaging of protoporphyrin IX (PpIX) fluorescence following topical application of 5-aminolevulinic acid (ALA). STUDY DESIGN/MATERIALS AND METHODS: Several hours after bladder instillation of an aqueous solution of ALA in 34 patients, a Krypton ion laser or a filtered Xenon arc-lamp was used to excite PpIX fluorescence. Tissue samples for histological analysis were taken while observing the bladder wall either by means of a video camera, or by direct endoscopic observation. RESULTS: A good correlation was found between the PpIX fluorescence and the histopathological diagnosis. On a total of 215 biopsies, 143 in fluorescent and 72 in nonfluorescent areas, all visible tumors on white light cytoscopy appeared in a bright red fluorescence with the photodetection technique. In addition, this method permitted to discover 47 unsuspected carcinomatous lesions on white light observation, among which 40% were carcinoma in situ. CONCLUSION: PpIX fluorescence induced by instillation into the bladder of 5-ALA is an efficient method of mapping the mucosa in bladder carcinoma.

Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology.

Human glandular kallikrein 2 (hK2) is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage-displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P'1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.

Hexyl-aminolevulinate mediated photodynamic therapy: how to spare normal urothelium. An in vitro approach

Résumé Objectifs : La thérapie photodynamique a pour but la destruction sélective du tissu néoplasique par interaction de lumière, d'oxygène et d'une substance photosensibilisatrice (la Protoporphyrine IX dans notre étude). Malgré une accumulation sélective du photosensibilisateur dans le tissu tumoral, la thérapie photodynamique du carcinome urothélial de la vessie peut endommager les cellules normales de l'épithélium urinaire. La prévention de ces lésions est importante pour la régénération de la muqueuse. Notre étude sur un modèle in vitro d'urothélium porcin étudie l'influence de la concentration du photosensibilisateur, des paramètres d'irradiation et de la production d'intermédiaires réactifs de l'oxygène (ROS) sur les effets photodynamique. Le but était de déterminer les conditions seuil pour épargner l'urothélium sain. Méthode: Dans une chambre de culture transparente à deux compartiments, des muqueuses vésicales de porc maintenues en vie ont été incubées avec une solution d'hexyl-aminolévulinate (HAL), le précurseur de la Protoporphyrine IX. Ces muqueuses ont ensuite été irradiées avec des doses lumineuses croissantes en lumière bleue et en lumière blanche, et les altérations cellulaires ont été évaluées par microscopie électronique à balayage et par un colorant fluorescent, le Sytox green. Nous avons également évalué la production d'intermédiaires réactifs de l'oxygène parla mesure de la fluorescence intracellulaire de Rhodamine 123 (R123), produit de l'oxydation de la Dihydrorhodamine 123 (DHR123) non fluorescente. Ces valeurs ont été corrélées avec celles du photo blanchiment de la PAIX. Résultats : Le taux de mortalité cellulaire était dépendant de la concentration de PAIX. Après 3 heures d'incubation, la valeur seuil de dose lumineuse pour la lumière bleu était de 0.15 et 0.75 J/cm2 (irradiance 30 et 75 mW/cm2, respectivement) et pour la lumière blanche de 0.55 J/cm2 (irradiante 30 mW/cm2). Le taux de photo blanchiment était inversement proportionnel à l'irradiante. Le système de détection des intermédiaires réactifs de l'oxygène DHR123/R123 a démontré une bonne corrélation avec les valeurs seuil pour toutes les conditions d'irradiation utilisées. Conclusions : Nous avons déterminé les doses lumineuses permettant d'épargner 50% des cellules urothéliales saines. L'utilisation d'une faible irradiante associée à des systèmes permettant de mesurer la production d'intermédiaires réactifs de l'oxygène dans les tissus irradiés pourrait améliorer la dosimétrie in vivo et l'efficacité de la thérapie photodynamique. Abstract Background and Objectives: Photodynamic therapy of superficial bladder cancer may cause damages to the normal surrounding bladder wall. Prevention of these is important for bladder healing. We studied the influence of photosensitizes concentration, irradiation parameters and production of reactive oxygen species (ROS) on the photodynamically induced damage in the porcine urothelium in vitro. The aim was to determine the threshold conditions for the cell survival. Methods: Living porcine bladder mucosae were incubated with solution of hexylester of 5-aminolevulinic acid (HAL). The mucosae were irradiated with increasing doses and cell alterations were evaluated by scanning electron microscopy and by Sytox green fluorescence. The urothelial survival score was correlated with Protoporphyrin IX (PpIX) photobleaching and intracellular fluorescence of Rhodamine 123 reflecting the ROS production. Results: The mortality ratio was dependent on PpIX concentration. After 3 hours of incubation, the threshold radiant exposures for blue light were 0.15 and 0.75 J/cm2 (irradiance 30 and 75 mW/cm2, respectively) and for white light 0.55 J/cm2 (irradiance 30 mW/cm2). Photobleaching rate increased with decreasing irradiance. Interestingly, the DHR123/R123 reporter system correlated well with the threshold exposures under all conditions used. Conclusions: we have determined radiant exposures sparing half of normal urothelial cells. We propose that the use of low irradiance combined with systems reporting the ROS production in the irradiated tissue could improve the in vivo dosimetry and optimize the PDT.

New human kallikrein 14: enzymatic characterization and inhibitors development.

RESUME DESTINE A UN LARGE PUBLIC En biologie, si une découverte permet de répondre à quelques questions, en général elle en engendre beaucoup d'autres. C'est ce qui s'est produit récemment dans le monde des kallicréines. De la famille des protéases, protéines ayant la faculté de couper plus ou moins spécifiquement d'autres protéines pour exercer un rôle biologique, la famille des kallicréines humaines n'était composée que de 3 membres lors du siècle dernier. Parmi eux, une kallicréine mondialement utilisée pour détecter le cancer de la prostate, le PSA. En 2000, un chercheur de l'hôpital universitaire Mont Sinaï à Toronto, le Professeur Eleftherios Diamandis, a découvert la présence de 12 nouveaux gènes appartenant à cette famille, situés sur le même chromosome que les 3 premières kallicréines. Cette découverte majeure a placé les spécialistes des kallicréines face à une montagne d'interrogations car les fonctions de ces nouvelles protéases étaient totalement inconnues. La kallicréine humaine 14 (hK14) présente un intérêt particulier, car elle se retrouve associée à différents cancers, notamment les carcinomes ovariens et mammaires. Cette association ne répond cependant pas à la fonction de cette protéase. L'objectif de ce travail de thèse était donc de découvrir, dans un premier temps, la spécificité de cette nouvelle kallicréine, c'est-à-dire le type de coupure qu'elle engendre au niveau des protéines qu'elle cible. Utilisant une technologie de pointe qui exploite la propriété des bactériophages à se répliquer dans les bactéries à l'infini, des dizaines de millions de combinaisons protéiques aléatoires ont été présentées à hK14, qui a pu sélectionner celles qui lui étaient favorables pour la coupure. Cette technique qualitative porte le nom de Phage Display Substrate. Une fois la sélection réalisée, il fallait transférer ces séquences coupées ou substrats dans un système permettant de donner une valeur quantitative à l'efficacité de coupure. Pour cela nous avons développé une technologie qui permet d'évaluer cette efficacité en utilisant des protéines fluorescentes de méduse, modifiées génétiquement, dont l'excitation de la première (CFP : cyan fluorescent protein) par la lumière à une certaine longue d'onde permet le transfert d'énergie à la seconde (YFP : yellow fluorescent protein), via un substrat qui les lie. Pour que ce transfert d'énergie se produise, il faut que les deux protéines fluorescentes soient proches, comme c'est le cas lorsqu'elles sont liées par un substrat. La coupure de ce lien provoque un changement de transfert d'énergie qui est quantifiable en utilisant un spectrofluoromètre. Cette technologie permet donc de suivre la réaction d'hydrolyse (coupure) des protéases. Afin de poursuivre certaines expériences permettant de mieux comprendre la fonction biologique d'hK14 ainsi que son éventuelle implication dans le cancer, nous avons développé des inhibiteurs spécifiques d'hK14. Les séquences qui on été le plus efficacement coupées par hK14 ont été utilisées pour transformer deux types d'inhibiteurs classiques, qui circulent dans notre sang, en inhibiteurs d'hK14 hautement efficaces et spécifiques. Selon les résultats obtenus in vitro, ils pourront être évalués in vivo en tant que traitement potentiel contre le cancer. RESUME Les protéases sont des enzymes impliquées dans des processus physiologiques mais aussi parfois pathologiques. La famille des kallicréines tissulaires humaines représente le plus grand groupe de protéases humaines, dont plusieurs pourraient participer au développement de certaines maladies. D'autre part, ces protéases sont apparues comme des marqueurs de pathogénicité potentiels, notamment dans les cas de cancers hormono-dépendants. La kallicréine humaine 14 a été récemment découverte et son implication dans quelques maladies, particulièrement dans le cas de tumeurs, semble probable. En effet, son expression génique est augmentée au niveau des tissus cancéreux de la prostate et du sein et son expression protéique s'est révélée plus élevée dans le sérum de patientes atteintes d'un cancer du sein ou des ovaires. Cependant, comme c'est le cas pour la plupart des kallicréines, sa fonction est encore inconnue. Afin de mieux connaître son rôle biologique et/ou pathologique, nous avons décidé de caractériser son activité enzymatique. Nous avons tout d'abord mis au point un système de substrats entièrement biologique permettant d'étudier in vitro l'activité des protéases. Ce système est basé sur le phénomène de FRET, à savoir le transfert d'énergie de résonance fluorescente qui intervient entre deux molécules fluorescentes voisines si le spectre d'émission de la protéine donneuse chevauche le spectre d'excitation de la protéine receveuse. Nous avons fusionné de manière covalente une protéine fluorescente bleue (CFP) et une jaune (YFP) en les liant avec diverses séquences. Par clivage de la séquence de liaison, une perte du transfert d'énergie peut être mesurée par un spectrofluoromètre. Cette technologie représente un moyen facile de suivre la réaction d'hydrolyse des protéases. Les conditions optimales de production de ces substrats CFP-YFP ont été déterminées, de même que les paramètres pouvant éventuellement influencer le FRET. Ce système possède une grande résistance à la protéolyse non spécifique et est applicable à un grand nombre de protéase. Contrairement aux substrats fluorogéniques, il permet d'étudier les acides aminés se trouvant des deux côtés du site de clivage. Ce système étant entièrement biologique, il est le reflet des interactions protéine-protéine et représente un outil biologique facile, bon marché et rapide pour caractériser les protéases. Dans un premier temps, hK14 a été mise en présence d' une banque de haute diversité de pentapeptides aléatoires présentée à la surface de phages afin d'identifier des substrats spécifiques. Ensuite, le système CFP-YFP a été employé pour trier les peptides sélectionnés afin d'identifier les séquences de substrats les plus sensibles et spécifiques pour hK14. Nous avons montré, qu'en plus de sa prévisible activité de type trypsine, hK14 possède aussi une très surprenante activité de type chymotrypsine. Les séquences les plus sensibles ont été choisies pour cribler la banque de donnée Swissprot, permettant ainsi l'identification de 6 substrats protéiques humains potentiels pour hK14. Trois d'entre eux, la laminine α-5, le collagène IV et la matriline-4, qui sont des composants de la matrice extracellulaire, ont démontré une grande susceptibilité à l'hydrolyse par hK14. De plus, la séparation éléctrophorétique a montré que la dégradation de la laminine α-5 et de la matriline-4 par hK14 devait se produire aux sites identifiés par la technologie du phage display. Pour terminer, nous avons transformé, par mutagenèse dirigée, deux serpines (inhibiteurs de protéases de type sérine) connues, AAT et ACT (alpha anti-trypsine et alpha anti-chymotrypsine), qui inhibent un vaste éventail d'enzymes humaines en inhibiteurs d'hK14 hautement efficaces et spécifiques. Ces inhibiteurs pourront être utilisés d'une part pour poursuivre certaines expériences permettant de mieux comprendre l'implication d'hK14 dans des voies physiologiques ou dans le cancer et d'autre part pour les évaluer in vivo en tant que traitement potentiel contre le cancer. SUMMARY Proteases consist of enzymes involved in physiological events, but also, in case of dysregulation, in pathogenicity. The human tissue kallikrein family represents the largest human protease cluster and includes several members that either could participate in the course of certain diseases or emerged as potential biological markers, especially in hormone dependent cancers. The human kallikrein 14 has been recently discovered and suggested implications in some disorders, particularly in tumors since its gene expression is up-regulated in prostate and breast cancer tissues and its protein expression increased in the serum of patients with breast and ovarian cancers. However, like most kallikreins, its function remains unknown. To better understand hK14 biological and/or pathological role, we decided to characterize its enzymatic activity. First of all, we developped a biological system suitable for in vitro study of protease activity. This system is based on the so-called FRET phenomenon, that is the Fluorescence Resonance Energy Transfer that occurs between two nearby fluorescent proteins if the emission spectrum of the donor overlaps the excitation spectrum of the acceptor. We fused covalently a cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) with diverses sequences. Upon cleavage of the linker sequence by protease, the loss of energy transfer can be measured by a spectrofluorometer allowing an easy following of hydrolysis reaction. The optimal conditions to produce in bacterial system these CFP-YFP substrates were determined as well as the parameters that could eventually influence the FRET. This system demonstrated a high degree of resistance to non-specific proteolysis and applicability to various conditions corresponding to a great number of existing proteases. Other avantages are the possibility to study the amino acids located both sides of the cleavage site as well as the interest to work in a full biological system reflecting protein-protein interaction. A phage substrate library with exhaustive diversity was used prior to CFP-substrate-YFP system to isolate specific human kallikrein 14 substrates. After that the CFP-YFP system was used to sort peptides and identify highly sensitive and specific substrate sequences for hK14. We showed that besides its predictable trypsin-like activity, hK14 also possesses a surprising chymotrypsin-like activity. The screening of the Swissprot database was achieved with the most sensitive sequences and allowed the identification of 6 potential human protein substrates for hK14. Three of them, laminin α-5, collagen IV and matrilin-4, which are components of the extracellular matrix were incubated with hK14, by which they were efficiently hydrolyzed. Moreover, electrophoretic separation revealed that degradation of laminin α-5 and matrilin-4 by hK14 generated fragments with identical molecular size than the predicted N-terminal fragments that would result from hK14 specific cleavage, proving the value of phage display substrate to identify potential substrates. Finally, with site-directed mutagenesis, we transformed two well-known serpins (serine protease inhibitors), AAT and ACT (alpha anti-trypsin and alpha anti-chymotrypsin), which inhibit a vast spectrum of human enzymes into highly efficient and specific hK14 inhibitors. These inhibitors will be used to pursue experiments that could help understand hK14 implication in physiological pathways as well as in cancer biology and also to perform their in vivo evalution as potential cancer treatment.

Intracellular excision and reintegration dynamics of the ICEclc genomic island of Pseudomonas knackmussii sp. strain B13.

Genomic islands are DNA elements acquired by horizontal gene transfer that are common to a large number of bacterial genomes, which can contribute specific adaptive functions, e.g. virulence, metabolic capacities or antibiotic resistances. Some genomic islands are still self-transferable and display an intricate life-style, reminiscent of both bacteriophages and conjugative plasmids. Here we studied the dynamical process of genomic island excision and intracellular reintegration using the integrative and conjugative element ICEclc from Pseudomonas knackmussii B13 as model. By using self-transfer of ICEclc from strain B13 to Pseudomonas putida and Cupriavidus necator as recipients, we show that ICEclc can target a number of different tRNA(Gly) genes in a bacterial genome, but only those which carry the GCC anticodon. Two conditional traps were designed for ICEclc based on the attR sequence, and we could show that ICEclc will insert with different frequencies in such traps producing brightly fluorescent cells. Starting from clonal primary transconjugants we demonstrate that ICEclc is excising and reintegrating at detectable frequencies, even in the absence of recipient. Recombination site analysis provided evidence to explain the characteristics of a larger number of genomic island insertions observed in a variety of strains, including Bordetella petri, Pseudomonas aeruginosa and Burkholderia.

Comparison of a modified shell vial culture procedure with conventional mouse inoculation for rabies virus isolation

Rabies is a neurotropic disease that is often lethal. The early diagnosis of rabies infection is important and requires methods that allow for the isolation of the virus from animals and humans. The present study compared a modified shell vial (MSV) procedure using 24-well tissue culture plates with the mouse inoculation test (MIT), which is considered the gold standard for rabies virus isolation. Thirty brain samples (25 positive and 5 negative by the fluorescent antibody test) obtained from different animal species at the National Institute of Hygiene Rafael Rangel in Caracas, Venezuela, were studied by the MIT and MSV assays. Nine samples (36%) were positive at 24 h, 10 (40%) were positive at 48 h and six (24%) were positive at 72 h by the MSV assay. With the MIT assay, 76% were positive at six days post inoculation and 12% were positive at 12 and 18 days post inoculation. One sample that was negative according to the MSV assay was positive with MIT on the 12th day. The MSV procedure exhibited a sensitivity of 96.2%, a specificity of 100%, a positive predictive value of 100% and a negative predictive value 80%. This procedure allowed for rapid rabies virus detection. MIT can be employed as an alternative method in laboratories without tissue culture facilities.

Analysis of Centrosome duplication in "Caenorhabditis elegans" : the role of "sas" genes

Abstract: The centrosome is the major microtubule organizing center (MTOC) of most animal cells. As such, it is essential for a number of processes, including polarized secretion or bipolar spindle assembly. Hence, centrosome number needs to be controlled precisely in coordination with DNA replication. Cells early in the cell cycle contain one centrosome that duplicates during S-phase to give rise to two centrosomes that organize a bipolar spindle during mitosis. A failure in this process is likely to engage the spindle assembly checkpoint and threaten genome stability. Despite its importance for normal and uncontrolled proliferation the mechanisms underlying centrosome duplication are still unclear. The Caenorhabditis elegans embryo is well suited to study the mechanisms of centrosome duplication. It allows for the analysis of cellular processes with high temporal and spatial resolution. Gene identification and inactivation techniques are very powerful and a wide set of mutant and transgenic strains facilitates analysis. My thesis project consisted of characterizing three sas-genes: sas-4, sas-5 and sas-¬6. Embryos lacking these genes fail to form a bipolar spindle, hence their name (spindle assembly). I established that sas-4(RNAi) and sas-6(RNAi) embryos do not form daughter centrioles and thus do not duplicate their centrosomes. Furthermore, I showed that both proteins localize to the cytoplasm and are strikingly enriched at centrioles throughout the cell cycle. By performing fluorescent recovery after photobleaching (FRAP) experiments and differentially labeling centrioles, I established that both proteins are recruited to centrioles once per cell cycle when daughter centrioles form. In contrast, SAS-5, PLK-1 and SPD-2 shuttle permanently between the cytoplasm and centrioles. By showing that SAS-5 and SAS-6 interact in vivo, I established a functional relationship between the proteins. Testing the putative human homologue of SAS-6 (HsSAS-6) and a distant relative of SAS-4 (CPAP), I was able to show that these proteins are required for centrosome duplication in human cells. In addition I found that overexpression of GFP¬HsSAS-6 leads to formation of extra centrosomes. In conclusion, we identified and gained important insights into proteins required for centrosome duplication in C. elegans and in human cells. Thus, our work contributes to further elucidate an important step of cell division in normal and malignant tissues. Eventually, this may allow for the development of novel diagnostic or therapeutic reagents to treat cancer patients. Résumé: Le centrosome est le principal centre organisateur des microtubules dans les cellules animales. De ce fait, il est essentiel pour un certain nombre de processus, comme l'adressage polarisé ou la mise en place d'un fuseau bipolaire. Le nombre de centrosome doit être contrôlé de façon précise et en coordination avec la réplication de l'ADN. Au début du cycle cellulaire, les cellules n'ont qu'un seul centrosome qui se duplique au cours de la phase S pour donner naissance à deux centrosomes qui forment le fuseau bipolaire pendant la mitose. Des défauts dans ce processus déclencheront probablement le "checkpoint" d'assemblage du fuseau et menaceront la stabilité du génome. Malgré leurs importances pour la prolifération normale ou incontrôlée des cellules, les mécanismes gouvernant la duplication des centrosomes restent obscures. L'embryon de Caenorhabditis elegans est bien adapté pour étudier les mécanismes de duplication des centrosomes. Il permet l'analyse des processus cellulaires avec une haute résolution spatiale et temporelle. L'identification des gènes et les techniques d'inactivation sont très puissantes et de larges collections de mutants et de lignées transgéniques facilitent les analyses. Mon projet de thèse a consisté à caractérisé trois gènes: sas-4, sas-5 et sas-6. Les embryons ne possédant pas ces gènes ne forment pas de fuseaux bipolaires, d'où leur nom (spindle assembly). J'ai établi que les embryons sas-4(RNAi) et sas-6(RNAi) ne forment pas de centrioles fils, et donc ne dupliquent pas leur centrosome. De plus, j'ai montré que les deux protéines sont localisées dans le cytoplasme et sont étonnamment enrichies aux centrioles tout le long du cycle cellulaire. En réalisant des expériences de FRAP (fluorscence recovery after photobleaching) et en marquant différentiellement les centrioles, j'ai établi que ces deux protéines sont recrutées une fois par cycle cellulaire aux centrioles, au moment de la duplication. Au contraire, SAS-5, PLK-1 et SPD-2 oscillent en permanence entre le cytoplasme et les centrioles. En montrant que SAS-5 et SAS-6 interagissent in vivo, j'ai établi une relation fonctionnelle entre les deux protéines. En testant les homologues humains putatifs de SAS-6 (HsSAS-6) et de SAS-4 (CPAP), j'ai été capable de montrer que ces protéines étaient aussi requises pour la duplication des centrosomes dans les cellules humaines. De plus, j'ai montré que la surexpression de GFP-HsSAS-6 entrainait la formation de centrosomes surnuméraires. En conclusion, nous avons identifié et progressé dans la compréhension de protéines requises pour la duplication des centrosomes chez C. elegans et dans les cellules humaines. Ainsi, notre travail contribue à mieux élucider une étape importante du la division cellulaire dans les cellules normales et malignes. A terme, ceci devrait aider au développement de nouveaux diagnostics ou de traitements thérapeuthiques pour soigner les malades du cancer.

DsRed2 transient expression in Culex quinquefasciatus mosquitoes

Culex quinquefasciatus mosquitoes have been successfully genetically modified only once, despite the efforts of several laboratories to transform and establish a stable strain. We have developed a transient gene expression method, in Culex, that delivers plasmid DNA directly to the mosquito haemolymph and additional tissues. We were able to express DsRed2 fluorescent protein in adult Cx. quinquefasciatus mosquitoes by injecting plasmids directly into their thorax. The expression of DsRed2 in adult Cx. quinquefasciatus mosquitoes is an important stepping stone to genetic transformation and the potential use of new control strategies and genetic interactions.

Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.

The microenvironment hosting a tumor actively participates in regulating tumor cell proliferation, migration, and invasion. Among the extracellular matrix proteins enriched in the stroma of carcinomas are the tenascin family members tenascin-C and tenascin-W. Whereas tenascin-C overexpression in gliomas is known to correlate with poor prognosis, the status of tenascin-W in brain tumors has not been investigated so far. In the present study, we analyzed protein levels of tenascin-W in 38 human gliomas and found expression of tenascin-W in 80% of the tumor samples, whereas no tenascin-W could be detected in control, nontumoral brain tissues. Double immunohistochemical staining of tenascin-W and von Willebrand factor revealed that tenascin-W is localized around blood vessels, exclusively in tumor samples. In vitro, the presence of tenascin-W increased the proportion of elongated human umbilical vein endothelial cells (HUVECs) and augmented the mean speed of cell migration. Furthermore, tenascin-W triggered sprouting of HUVEC spheroids to a similar extent as the proangiogenic factor tenascin-C. In conclusion, our study identifies tenascin-W as a candidate biomarker for brain tumor angiogenesis that could be used as a molecular target for therapy irrespective of the glioma subtype.-Martina, E., Degen, M., Rüegg, C., Merlo, A., Lino, M. M., Chiquet-Ehrismann, R., Brellier, F. Tenascin-W is a specific marker of glioma-associated blood vessels and stimulates angiogenesis in vitro.