Modifications de la matrice extracellulaire dans la rigidité artérielle

La paroi vasculaire est composée de cellules endothéliales, de cellules musculaires lisses vasculaires et de fibroblastes qui sont entourés d’un réseau structuré et complexe de protéines, la matrice extracellulaire. Les interactions réciproques entre la matrice et les cellules sont nécessaires à la croissance, au développement et au remodelage. Or, différents contextes pathologiques entraînent la perturbation de ces interactions et sont la cause de différentes maladies. Au cours du vieillissement, la matrice extracellulaire des grosses artères élastiques est modifiée. Ainsi, les lamelles élastiques de la paroi vasculaire se fragmentent ou sont dégradées, en plus de calcifier. De même, l’accumulation de protéines plus rigides, comme le collagène, entraîne le développement de la fibrose. Ces modulations vont mener à l’augmentation de la rigidité artérielle et au développement de l’hypertension systolique isolée. En utilisant un modèle animal de calcification basé sur l’inhibition d’une protéine anti-calcifiante, la matrix Gla protein, avec la warfarine, nous avons étudié la séquence des événements impliqués dans le développement de l’hypertension systolique isolée. Nous avons observé l’activation précoce et transitoire de MMP-9, puis du TGF-ß, précédant la modulation phénotypique des cellules musculaires lisses vasculaires, la calcification et les changements hémodynamiques. L’inhibition des métalloprotéinases et du TGF-ß a permis de prévenir la calcification vasculaire. Nous avons également étudié le rôle joué par une enzyme de la matrice extracellulaire, la transglutaminase 2, dans le développement de la calcification associée à l’hypertension systolique isolée. À l’aide d’un nouvel inhibiteur de cette enzyme, qui a permis de prévenir la calcification, nous avons établi que la transglutaminase était un élément clé dans le processus pathologique. Ces travaux ont permis de démontré l’intérêt de nouvelles avenues thérapeutiques ciblant directement la matrice extracellulaire, particulièrement la MMP-9, le TGF-ß et la transglutaminase 2, dans la pathologie de l’hypertension systolique isolée.

La dihydrofolate réductase R67, comme une cible d’antibiotiques et biocatalyseur potentiel

La dihyrofolate réductase de type II R67 (DHFR R67) est une enzyme bactérienne encodée par un plasmide donc aisément transmissible. Elle catalyse la réaction de réduction du dihydrofolate (DHF) en tétrahydrofolate (THFA) essentiel pour la prolifération cellulaire. La DHFR R67 est une enzyme qui dépend du cofacteur NADPH. La DHFR R67 est différente, structurellement et génétiquement, de l’enzyme DHFR chromosomale présente chez tous les organismes et elle est résistante au triméthoprime (TMP) qui est largement utilisé dans les traitements antibactériens chez l’Homme. Aucun inhibiteur sélectif contre la DHFR R67 n’est actuellement répertorié. Le but de cette étude a été d’identifier des molécules qui pourront inhiber la DHFR R67 sélectivement, sans affecter la DHFR humaine (DHFRh). La vérification de la qualité des essais enzymatiques en conditions déterminées pour le criblage d’inhibiteurs sur plusieurs lectrices à plaques a identifié des appareils appropriés pour l’analyse. L’étude de l’activité enzymatique de la DHFR R67 et de la DHFRh en présence des solvants organiques et liquides ioniques (LIs), comme des co-solvants pour le criblage rationnel d’inhibiteurs, a montré que certains LIs peuvent servir de milieu alternatif pour les essais enzymatiques. Le criblage rationnel basé sur l’approche du design d’un inhibiteur à partir de petites molécules, a révélé des molécules primaires qui inhibent la DHFR R67 de façon faible, mais sélective. Le test des composés biologiquement actifs qui comprennent des petits fragments, a montré l’augmentation de l’affinité entre la DHFR R67 et les composés testés. Trois composés ont été déterminés comme des inhibiteurs sélectifs prometteurs pour la DHFR R67.

Land surface temperature retrieval from Sentinel 2 and 3 Missions

In this work we explore the synergistic use of future MSI instrument on board Sentinel-2 platform and OLCI/SLSTR instruments on board Sentinel-3 platform in order to improve LST products currently derived from the single AATSR instrument on board the ENVI- SAT satellite. For this purpose, the high spatial resolu- tion data from Setinel2/MSI will be used for a good characterization of the land surface sub-pixel heteroge- neity, in particular for a precise parameterization of surface emissivity using a land cover map and spectral mixture techniques. On the other hand, the high spectral resolution of OLCI instrument, suitable for a better characterization of the atmosphere, along with the dual- view available in the SLTSR instrument, will allow a better atmospheric correction through improved aero- sol/water vapor content retrievals and the implementa- tion of novel cloud screening procedures. Effective emissivity and atmospheric corrections will allow accu- rate LST retrievals using the SLSTR thermal bands by developing a synergistic split-window/dual-angle algo- rithm. ENVISAT MERIS and AATSR instruments and different high spatial resolution data (Landsat/TM, Proba/CHRIS, Terra/ASTER) will be used as bench- mark for the future OLCI, SLSTR and MSI instruments. Results will be validated using ground data collected in the framework of different field campaigns organized by ESA.

Interferon-α antagonizes IL-3-mediated immunoregulatory functions of human basophils

Human basophils are major inflammatory cells in maintaining chronic allergic asthma. It has been published that interferon-α (IFN-α) improves clinical symptoms of asthma patients. In contrast, IL-3 exacerbates airway inflammation by inducing IL-4, IL-8 and IL-13 secretion from human basophils thus regulating their immunoregulatory functions. Furthermore, IL-3 exceptionally promotes survival of basophils. Here, we assessed cellular response of human basophils treated with IFN-α alone or in combination with IL-3. Our data show that IFN-α enhances apoptosis in purified human blood basophils compared to spontaneous apoptosis of controls or IFN-γ treated cells. Furthermore, we demonstrate that both IFN-α and FasL enhance apoptosis in human basophils with similar efficiency in a rather additive than synergistic way. IFN-α inhibits IL-3-induced survival to a minor degree. Particularly however, it suppresses IL-3-induced de-novo production of IL-8 and IL-13 up to 80%. In contrast, the production of IL-4 is not affected. Analyses of signaling pathways reveal that IFN-α promotes prolonged phosphorylation of STAT1/STAT2. By using a pan-JAK inhibitor the phosphorylation of STAT1/STAT2 is inhibited and most importantly the pro-apoptotic effect of IFN-α is abolished. Although the phosphorylation of p38-MAPK in IFN-α-treated cells is comparable to non-treated cells, inhibition of p-p38 activity abrogates IFN-α-enhanced apoptosis as well. We conclude that IFN-α-enhanced apoptosis is tightly regulated by the cooperation of JAK/STAT and p38-MAPK pathways. Our study identifies IFN-α as a novel inhibitor of IL-3-induced IL-8 and IL-13 production of human basophils. Taken together our study may explain the improved clinical symptoms of asthma patients treated with IFN-α.

Emulation of random output simulators

Computer models, or simulators, are widely used in a range of scientific fields to aid understanding of the processes involved and make predictions. Such simulators are often computationally demanding and are thus not amenable to statistical analysis. Emulators provide a statistical approximation, or surrogate, for the simulators accounting for the additional approximation uncertainty. This thesis develops a novel sequential screening method to reduce the set of simulator variables considered during emulation. This screening method is shown to require fewer simulator evaluations than existing approaches. Utilising the lower dimensional active variable set simplifies subsequent emulation analysis. For random output, or stochastic, simulators the output dispersion, and thus variance, is typically a function of the inputs. This work extends the emulator framework to account for such heteroscedasticity by constructing two new heteroscedastic Gaussian process representations and proposes an experimental design technique to optimally learn the model parameters. The design criterion is an extension of Fisher information to heteroscedastic variance models. Replicated observations are efficiently handled in both the design and model inference stages. Through a series of simulation experiments on both synthetic and real world simulators, the emulators inferred on optimal designs with replicated observations are shown to outperform equivalent models inferred on space-filling replicate-free designs in terms of both model parameter uncertainty and predictive variance.

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing >= 50% inhibition property against CHIKV at 10 mu M were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC50 values between 2.2 mu M and 7.1 mu M. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity -inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta -bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor. (c) 2007 Elsevier Inc. All rights reserved.

Functional peptidomics of amphibian skin secretion: A novel Kunitz-type chymotrypsin inhibitor from the African hyperoliid frog, Kassina senegalensis

Amphibian skin secretions are, for the most part, complex peptidomes. While many peptide components have been biologically- and structurally-characterised into discrete "families", some of which are analogues of endogenous vertebrate regulatory peptides, a substantial number are of unique structure and unknown function. Among the components of these secretory peptidomes is an array of protease inhibitors. Inhibitors of trypsin are of widespread occurrence in different taxa and are representative of many established structural classes, including Kunitz, Kazal and Bowman-Birk. However, few protease inhibitors with activity against other specific proteases have been described from this source. Here we report for the first time, the isolation and structural characterisation of an inhibitor of chymotrypsin of Kunitz-type from the skin secretion of the African hyperoliid frog, Kassina senegalensis. To this end, we employed a functional peptidomic approach. This scheme involves fractionation of the peptidome, functional end-point screening, structural characterisation of resultant actives followed by molecular cloning of biosynthetic precursor-encoding cDNA(s). The novel mature and active polypeptide identified consisted of 62 amino acid residues (average molecular mass 6776.24 Da), of which 6 were positionally-conserved cysteines. The P(1) position within the active site was occupied by a phenylalanyl residue. Bioinformatic analysis of the sequence using BLAST, revealed a structural similarity to Kunitz-type chymotrypsin inhibitors from other organisms, ranging from silkworms to snakes.

Targeting low-druggability bromodomains: fragment based screening and inhibitor design against the BAZ2B bromodomain

Bromodomains are epigenetic reader domains that have recently become popular targets. In contrast to BET bromodomains, which have proven druggable, bromodomains from other regions of the phylogenetic tree have shallower pockets. We describe successful targeting of the challenging BAZ2B bromodomain using biophysical fragment screening and structure-based optimization of high ligand-efficiency fragments into a novel series of low-micromolar inhibitors. Our results provide attractive leads for development of BAZ2B chemical probes and indicate the whole family may be tractable.

RNA interference screening identifies a novel role for PCTK1/CDK16 in medulloblastoma with c-Myc amplification.

Medulloblastoma (MB) is the most common malignant brain tumor in children and is associated with a poor outcome. cMYC amplification characterizes a subgroup of MB with very poor prognosis. However, there exist so far no targeted therapies for the subgroup of MB with cMYC amplification. Here we used kinome-wide RNA interference screening to identify novel kinases that may be targeted to inhibit the proliferation of c-Myc-overexpressing MB. The RNAi screen identified a set of 5 genes that could be targeted to selectively impair the proliferation of c-Myc-overexpressing MB cell lines: AKAP12 (A-kinase anchor protein), CSNK1α1 (casein kinase 1, alpha 1), EPHA7 (EPH receptor A7) and PCTK1 (PCTAIRE protein kinase 1). When using RNAi and a pharmacological inhibitor selective for PCTK1, we could show that this kinase plays a crucial role in the proliferation of MB cell lines and the activation of the mammalian target of rapamycin (mTOR) pathway. In addition, pharmacological PCTK1 inhibition reduced the expression levels of c-Myc. Finally, targeting PCTK1 selectively impaired the tumor growth of c-Myc-overexpressing MB cells in vivo. Together our data uncover a novel and crucial role for PCTK1 in the proliferation and survival of MB characterized by cMYC amplification.

Discovery and characterization of a novel non-competitive inhibitor of the divalent metal transporter DMT1/SLC11A2

Divalent metal transporter-1 (SLC11A2/DMT1) uses the H+ electrochemical gradient as the driving force to transport divalent metal ions such as Fe2+, Mn2+ and others metals into mammalian cells. DMT1 is ubiquitously expressed, most notably in proximal duodenum, immature erythroid cells, brain and kidney. This transporter mediates H+-coupled transport of ferrous iron across the apical membrane of enterocytes. In addition, in cells such as to erythroid precursors, following transferrin receptor (TfR) mediated endocytosis; it mediates H+-coupled exit of ferrous iron from endocytic vesicles into the cytosol. Dysfunction of human DMT1 is associated with several pathologies such as iron deficiency anemia hemochromatosis, Parkinson's disease and Alzheimer's disease, as well as colorectal cancer and esophageal adenocarcinoma, making DMT1 an attractive target for drug discovery. In the present study, we performed a ligand-based virtual screening of the Princeton database (700,000 commercially available compounds) to search for pharmacophore shape analogs of recently reported DMT1 inhibitors. We discovered a new compound, named pyrimidinone 8, which mediates a reversible linear non-competitive inhibition of human DMT1 (hDMT1) transport activity with a Ki of ∼20 μM. This compound does not affect hDMT1 cell surface expression and shows no dependence on extracellular pH. To our knowledge, this is the first experimental evidence that hDMT1 can be allosterically modulated by pharmacological agents. Pyrimidinone 8 represents a novel versatile tool compound and it may serve as a lead structure for the development of therapeutic compounds for pre-clinical assessment.

Development of HIF-1α/HIF-1β heterodimerization inhibitors using a novel bioluminescence reporter assay system for in vitro high throughput screening and in vivo imaging

Tumor growth often outpaces its vascularization, leading to development of a hypoxic tumor microenvironment. In response, an intracellular hypoxia survival pathway is initiated by heterodimerization of hypoxia-inducible factor (HIF)-1α and HIF-1β, which subsequently upregulates the expression of several hypoxia-inducible genes, promotes cell survival and stimulates angiogenesis in the oxygen-deprived environment. Hypoxic tumor regions are often associated with resistance to various classes of radio- or chemotherapeutic agents. Therefore, development of HIF-1α/β heterodimerization inhibitors may provide a novel approach to anti-cancer therapy. To this end, a novel approach for imaging HIF-1α/β heterodimerization in vitro and in vivo was developed in this study. Using this screening platform, we identified a promising lead candidate and further chemically derivatized the lead candidate to assess the structure-activity relationship (SAR). The most effective first generation drug inhibitors were selected and their pharmacodynamics and anti-tumor efficacy in vivo were verified by bioluminescence imaging (BLI) of HIF-1α/β heterodimerization in the xenograft tumor model. Furthermore, the first generation drug inhibitors, M-TMCP and D-TMCP, demonstrated efficacy as monotherapies, resulting in tumor growth inhibition via disruption of HIF-1 signaling-mediated tumor stromal neoangiogenesis.

A novel DNA damage inducible inhibitor of p53

p53 is required for the maintenance of the genomic stability of cells. Mutations in the p53 tumor-suppressor gene occur in more than 50% of human cancers of diverse types. In addition, 70% of families with Li-Fraumeni syndrome have a germline mutation in p53, predisposing these individuals to multiple forms of cancer. In response to DNA damage, p53 becomes stabilized and activated. However the exact mechanism by which DNA damage signals the stabilization and activation of p53 still remains elusive. The biochemical activity of p53 that is required for tumor suppression, and presumably the cellular response to DNA damage, involves the ability of the protein to bind to specific DNA sequences and to function as a transcription factor. For the downstream targets, p53 transactivates many genes involved in growth arrest, apoptosis and DNA repair such as p21, Bax and GADD45, respectively. An open question in the field is how cells can determine the downstream effects of p53. ^ We hypothesize that, through its associated proteins, p53 can differentially transactivate its target genes, which determine its downstream effect. Additionally, p53 interacting proteins may be involved in signaling for the stabilization and activation of p53. Therefore, a key aspect to understanding p53 function is the identification and analysis of proteins that interact with it. We have employed the Sos recruitment system (SRS), a cytoplasmic yeast two-hybrid screen to identify p53 interacting proteins. The SRS is based on the ability of Sos to activate Ras when it becomes localized to the plasma membrane. The system takes advantage of an S. cerevisiae strain, cdc25-2 temperature sensitive mutant, harboring a mutation in Sos. In this strain, fusion proteins containing a truncated Sos will only localize to the membrane by protein-protein interaction, which allows growth at non-permissive temperature. This system allows the use of intact transcriptional activators such as p53. ^ To date, using a modified SRS library screen to identify p53 interacting proteins, I have identified p53 (known to interact with itself) and a novel p53-interacting protein (PIP). PIP is a specific p53 interacting protein in the SRS. The interaction of p53 and PIP was further confirmed by performing in vitro and in vivo binding assays. In the in vivo binding study, the interaction can only be detected in the presence of ionizing radiation suggesting that this interaction might be involved in DNA-damage induced p53-signalling pathway. After screening cDNA and genomic libraries, a full-length PIP-cDNA clone ( ∼ 3kb) was obtained which encodes a protein of 429 amino acids with calculated molecular weight of 46 kDa. The results of genebank search indicated that the PIP is an unidentified gene and contains a conserved ring-finger domain, which is present in a diverse family of regulatory proteins involved in different aspects of cellular function. Northern blot analysis revealed that the size of its messenge is approximately 3 kb preferentially expressed in brain, heart, liver and kidney. The PIP protein is mainly located in the cytoplasm as determined by the cellular localization of a green fluorescence fusion protein. Preliminary functional analysis revealed that PIP downregulated the transactivation activity of p53 on both p21 and mdm2 promoters. Thus, PIP may be a novel negative regulator of p53 subsequent to DNA damage. ^

A novel, automated nutrition screening system as a predictor of nutritional risk in an oncology day treatment unit (ODTU)

Purpose Paper-based nutrition screening tools can be challenging to implement in the ambulatory oncology setting. The aim of this study was to determine the validity of the Malnutrition Screening Tool (MST) and a novel, automated nutrition screening system compared to a ‘gold standard’ full nutrition assessment using the Patient-Generated Subjective Global Assessment (PG-SGA). Methods An observational, cross-sectional study was conducted in an outpatient oncology day treatment unit (ODTU) within an Australian tertiary health service. Eligibility criteria were as follows: ≥18 years, receiving outpatient anticancer treatment and English literate. Patients self-administered the MST. A dietitian assessed nutritional status using the PGSGA, blinded to the MST score. Automated screening system data were extracted from an electronic oncology prescribing system. This system used weight loss over 3 to 6 weeks prior to the most recent weight record or age-categorised body mass index (BMI) to identify nutritional risk. Sensitivity and specificity against PG-SGA (malnutrition) were calculated using contingency tables and receiver operating curves. Results There were a total of 300 oncology outpatients (51.7 % male, 58.6±13.3 years). The area under the curve (AUC) for weight loss alone was 0.69 with a cut-off value of ≥1 % weight loss yielding 63 % sensitivity and 76.7 % specificity. MST (score ≥2) resulted in 70.6 % sensitivity and 69.5 % specificity, AUC 0.77. Conclusions Both the MST and the automated method fell short of the accepted professional standard for sensitivity (~≥80 %) derived from the PG-SGA. Further investigation into other automated nutrition screening options and the most appropriate parameters available electronically is warranted to support targeted service provision.