I. Identification and characterisation of epigenetically altered tumor supressor genes by proteomic and genomic approches / II. Studies of genes regualted by MeCP2 union to its promoter regions by proteomic and genmomic approches

DNA methylation has an important impact on normal cell physiology, thus any defects in this mechanism may be related to the development of various diseases In this project we are interested in identifying epigeneticaliy modified genes, in general controlled by processes related to the DNA methylation, by means of a new strategy combining protomic and genomic analyses. First, the two Dimensional-Difference Gel Electrophoresis (2-DIGE) protein analyses of extracts obtained from HCT-116 wt and double knockout for DNMT1 and DNMT3b (DKO) cells revealed 34 proteins overexpressed in the condition of DNMTs depletion. From five genes with higher transcript lavels in DKO cells, comparing with HCT-116 wt. oniy AKR1B1, UCHLl and VIM are melhylated in HCT-116. As expected. the DNA methvlation 1s lost in DKO cells. The rneth,vl ation of VIM and UCHLl promoters in some cancer samples has already been repaired, thus further studies has been focused on AKRlBI. AKR1B1 expression due lo DNA methyiaton of promoter region seems to occur specilfically in the colon cancer cell Iines. which was confirmed in the DNA rnethylation status and expression analyses. performed on 32 different cancer cell lines (including colon, breast, lymphoma, leukemia, neuroblastoma, glioma and lung cancer cell Iines) as well as normal colon and normal lymphocytes samples. AKRIBI expression after treatments with DNA demethvlating agent (AZA) was rescued in 5 coloncancer cell lines (including genetic regulation of the candidate gene. The methylation status of the rest of the genes identified in proteomic analysis was checked by methylation specific PCR (MSP) experiment and all appeared to be unmethylated. The similar research has been done also bv means of Mecp2-null mouse model For 14 selected candidate genes the analyses of expression leveis, methylation Status and MeCP2 interaction with promoters are currently being performed.

Serological diagnosis of Chagas disease: evaluation and characterisation of a low cost antigen with high sensitivity and specificity

In spite of evident progress in the serology of Chagas disease, the requirement for new diagnostic antigens persists. We have evaluated different antigens obtained from Trypanosoma cruzi grown in medium rich in nutrients or under nutrient stress, autoclaved or sonicated and fractionated by differential centrifugation. The resulting antigens were evaluated for diagnosis of Chagas disease using ELISA. Immunofluorescence of the parasites demonstrated that nutrient stress induced changes in the distribution and density of antigens recognised by a pool of sera from experimentally infected mice. When evaluated using ELISA, it was evident that most fractions had good sensitivity but poor specificity. Surprisingly, the best specificity and sensitivity was observed with parasites cultured under nutrient stress and autoclaved. Furthermore this antigen had low cross reactivity with sera from other parasitic diseases, Leishmaniasis in particular. Western blot analysis demonstrated that autoclaving seems to non-specifically eliminate cross-reactive antigens. In conclusion, autoclaving epimastigotes of T. cruzi, after nutrient stress, allowed us to obtain an antigen that could be used in the serological diagnosis of Chagas disease.

Cloning, expression and characterisation of an HtrA-like serine protease produced in vivo by Mycobacterium leprae

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.

Synthesis and transport of creatine in the CNS: importance for cerebral functions.

J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06935.x Abstract Apart of its well known function of 'energetic buffer' through the creatine/phosphocreatine/creatine kinase system allowing the regeneration of ATP, creatine has been recently suggested as a potential neuromodulator of even true neurotransmitter. Moreover, the recent discovery of primary creatine deficiency syndromes, due to deficiencies in l-arginine : glycine amidinotransferase or guanidinoacetate methyltransferase (the two enzymes allowing creatine synthesis) or in the creatine transporter, has shed new light on creatine synthesis, metabolism and transport, in particular in CNS which appears as the main tissue affected by these creatine deficiencies. Recent data suggest that creatine can cross blood-brain barrier but only with a poor efficiency, and that the brain must ensure parts of its needs in creatine by its own endogenous synthesis. Finally, the recent years have demonstrated the interest to use creatine as a neuroprotective agent in a growing number of neurodegenerative diseases, including Parkinson's and Huntington's diseases. This article aims at reviewing the latest data on creatine metabolism and transport in the brain, in relation to creatine deficiencies and to the potential use of creatine as neuroprotective molecule. Emphasis is also given to the importance of creatine for cerebral function.

Emergence and characterisation of vanB vancomycin-resistant Enterococcus faecalis in Rio de Janeiro, Brazil

Here we describe the detection and characterisation of three isolates of vancomycin-resistant VanB-type Enterococcus faecalis. Sequence analysis suggested that these isolates harboured the vanB1 gene. The isolates were susceptible to the majority of antimicrobial agents tested, with the exception of chloramphenicol, erythromycin and vancomycin, and showed distinct profiles of high-level resistance to aminoglycosides. Analysis of the clonal relatedness of the vanB E. faecalis isolates showed similar pulsed-field gel electrophoresis profiles. To our knowledge, this is the first report of the occurrence of enterococcal strains carrying vanB genes in Brazil.

Identification and characterisation of microRNAs in young adults of Angiostrongylus cantonensis via a deep-sequencing approach

Angiostrongylus cantonensis is an important causative agent of eosinophilic meningitis and eosinophilic meningoencephalitis in humans. MicroRNAs (miRNAs) are small non-coding RNAs that participate in a wide range of biological processes. This study employed a deep-sequencing approach to study miRNAs from young adults of A. cantonensis. Based on 16,880,456 high-quality reads, 252 conserved mature miRNAs including 10 antisense miRNAs that belonging to 90 families, together with 10 antisense miRNAs were identified and characterised. Among these sequences, 53 miRNAs from 25 families displayed 50 or more reads. The conserved miRNA families were divided into four groups according to their phylogenetic distribution and a total of nine families without any members showing homology to other nematodes or adult worms were identified. Stem-loop real-time polymerase chain reaction analysis of aca-miR-1-1 and aca-miR-71-1 demonstrated that their level of expression increased dramatically from infective larvae to young adults and then decreased in adult worms, with the male worms exhibiting significantly higher levels of expression than female worms. These findings provide information related to the regulation of gene expression during the growth, development and pathogenesis of young adults of A. cantonensis.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.

Synthesis and characterization of kraft lignin-based epoxy resins

Epoxidization is an interesting way to develop a new application of lignin and therefore to improve its application potential. In this work, kraft lignin-based epoxy resins were obtained by the epoxidization reaction, using the kraft lignin recovered directly from pulping liquor and modified by a methylolation reaction. The methylolated lignins were obtained by the reaction of original kraft lignin with formaldehyde and glyoxal, which is a less volatile and less toxic aldehyde. 1H-NMR spectroscopy showed that methylolated kraft lignin has more hydroxymethyl groups than glyoxalated kraft lignin. For the epoxidization reaction we studied the influence of the lignin:NaOH (w/w) ratio, temperature, and time of the reaction on the properties of the prepared epoxidized lignins. The structures of lignin-based epoxy resins were followed by epoxy index test and FTIR spectroscopy. Optimal conditions were obtained for lignin-based epoxy resin produced at lignin/NaOH = 1/3 at 70 ºC for 3h. Thermogravimetry analysis (TGA) revealed that the epoxidization enhances the thermal stability of lignins and may allow a wider temperature range for applications with lignin epoxy-PF blends

Experimental and computational study on the synthesis and characterization of self-assembling meso/macroporous materials in highly concentrated emulsions

Report for the scientific sojourn carried out at Massachusetts General Hospital Cancer Center-Harvard Medical School, Estats Units, from 2010 to 2011. The project aims to study the aggregation behavior of amphiphilic molecules in the continuous phase of highly concentrated emulsions, which can be used as templates for the synthesis of meso/macroporous materials. At this stage of the project, we have investigated the self-assembly of diblock and triblock surfactants under the effect of a confined geometry being surrounded by the droplets of the dispersed phase. These droplets limit the growth of the aggregates, deeply modify their orientation and hence alter their spatial arrangement as compared to the self-assembly taking place far enough from any boundary surface, that is in the bulk. By performing Monte Carlo simulations, we have showed that the interface between the dispersed and continuous phases as well as its shape has a significant impact on the structural order of the resulting aggregates and hence on the potential applications of highly concentrated emulsions as reaction media, drug delivery systems, or templates for meso/macroporous materials. Due to the combined effect of symmetry breaking and morphological frustration, very intriguing structures, such as square columnar liquid crystals, twisted X-shaped aggregates, and helical phases of cylindrical aggregates, never observed in the bulk for the same model surfactant, have been found. The presence of other more conventional structures, such as micelles and cubic and hexagonal liquid crystals, formed at low and high amphiphilic concentrations, respectively, further enhance the interest on this already rich aggregation behavior.

Gene expression profiling of human glioblastomas for the identification of predictive factors and characterisation of molecular mechanism implicated in response to therapy and outcome

ABSTRACT Poor outcome for glioblastoma patients is largely due to resistance to chemoradiation therapy. While epigenetic inactivation of MGMT mediated DNA repair is highly predictive for benefit from the alkylating agent therapy Temozolomide, additional mechanisms for resistance associated with molecular alterations exist. Furthermore, new concepts in cancer suggest that resistance to treatment may be linked to cancer stem cells that escape therapy and act as source for tumour recurrence. We determined gene expression signatures associated with outcome in glioblastoma patients enrolled in a phase II and phase III clinical trial establishing the new combination therapy of radiation plus concomitant and adjuvant Temozolomide. Correlating stable gene clusters emerging from unsupervised analysis with survival of 42 treated patients identified a number of biological processes associated with outcome. Most prominent, a gene cluster dominated by HOX genes and comprising PROM1, was associated with resistance. PROM1 encodes CD133, a marker for a subpopulation of tumour cells enriched for glioblastoma stem- like cells. The core of this correlated HOX cluster was comprised in the top genes of a "self-renewal signature" defined in a mouse model for MLL-AF9 initiated leukaemia. The association of the HOX gene cluster with tumour resistance was confirmed in two external data sets of 146 malignant glioma As additional resistance factors we identified over-expression of the epidermal growth factor receptor gene, EGFR, while increased gene expression related to biological features of tumour host interaction, including markers for tumour vascular and cell adhesion, and innate immune response, were associated with better outcome. The "self-renewal" signature associated with resistance to the new combination chemoradiation therapy provides first clinical evidence that glioma stem like cells may implicated in resistance in a uniformly treated cohort of glioblastoma patients. This study underlines the need to target the tumour stem cell compartment, and provides some testable hypothesis for biological mechanisms relevant for malignant behaviour of glioblastoma that may be targeted in new treatment approaches. Résumé Le glioblastome, tumeur cérébrale primaire maligne la plus fréquente, est connue pour son mauvais pronostique. Des avancées chimiothérapeutiques récentes avec des agents alkylants comme le témozolomide (TMZ), ont permis une amélioration notable dans la survie de certains patients. Les bénéficiaires ont la caractéristique commune de présenter une particularité génétique, la methylation du MGMT (methylguanine methyltransferase). Néanmoins, d'autres mécanismes de résistance en fonction des aberrations moléculaires existent. Nous avons établi les profils d'expressions génétiques des patients traités par irradiation et TMZ dans des études cliniques de phase II et III. En combinant des méthodes non-supervisées et supervisées, de l'étude de la cohorte des patients traités nous avons découvert des groupes de gènes associés à la survie. Un ensemble de gènes contenant les gènes Hox semble lié au mécanisme de résistance au traitement. Récemment, les gènes Hox ont été décrits comme faisant partie d"une signature d'autorenouvellement (self-renewal) des cellules souches cancéreuses de la leucémie. L'autorenouvellement est un processus grâce auquel les cellules souches se maintiennent tout au long de la vie. Cette association à la résistance est confirmée dans deux autres études indépendantes. Un autre facteur de résistance au traitement est la surexpression du gène EGFR. D'autre part, deux groupes de gènes associés à la relation entre hôte-tumeur tels que les marqueurs des vaisseaux tumoraux et de la réponse immunitaire innée s'avèrent avoir un effet positif sur la survie des patients traités. La découverte de la signature d'autorenouvellement comme facteur de résistance à la nouvelle chimio-radiothérapie offre une preuve clinique que les cellules souches cancéreuses sont impliquées dans la résistance au traitement. If est donc logique de penser que le traitement ciblé contre des cellules souches cancéreuses va dans l'avenir permettre des thérapies anticancéreuses plus performantes.

On the Source and Rewriting of 1 Corinthians 2.9 in Christian, Jewish and Islamic Traditions (1 Clem 34.8; GosJud 47.10-13; a ḥadīth qudsī)

The article reopens the file of sources, parallels and rewritings of 1 Cor 2.9, a saying that Paul attributes to some written source, when others sources put it into Jesus' mouth (e.g. GosThom 17). A state of research highlights that the hypothesis of an oral source is generally preferred but an accurate study of 1 Clem 34.8, a parallel too often neglected, supports the presence of a written source that existed before 1 Cor 2.9. GosJud 47.10-13 will help to understand the attribution of the saying to Jesus. The last important part of this article studies its parallel in Islamic traditions, a ḥadīth qudsī.

Isolation, culture and characterisation of human endothelial progenitor cells

Temps de parole: 30 minutes

Calcium phosphate biomaterials from marine algae. Hydrothermal synthesis and characterisation

Calcium phosphate compounds such as Hydroxyapatite (HAp) were prepared by hydrothermal synthesis with phycogenic CaCO3 as starting material. Material obtained was characterised by usual methods (XRD, FTIR, TG, N2-adsorption, SEM and EDX) in order to study its physical-chemical characteristics. The prepared HAp showed that it may be suitable for use as a biomaterial.

Synthesis and characterization of oxovanadium (IV) dithiocarbamates with pyridine

We report the synthesis and study of a new series of oxovanadium (IV) dithiocarbamate adducts and derivatives with pyridine and cyclohexyl, di-iso-butyl, di-n-propyl, anilin, morpholin, piperidin and di-iso-propyl amines. The complexes have been characterized by analytical, magnetochemical, IR, visible-UV spectral and thermal studies, and are assigned the formulas [VO(L)2].py, where L=cyclohexyl, di-iso-butyl, di-n-propyl, anilin dithiocarbamate and [VO(OH)(L)(py)2]OH.H2O (L=morpholin, piperidin and di-iso-propyl dithiocarbamate). The effect of the adduct formation on the pV=0 bound is discussed in terms of the IR (V=O, V-S and V-N stretching frequencies) and electronic spectra (d-d transitions).