Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Spectral retinal image processing and analysis for ophthalmology

Diabetic retinopathy, age-related macular degeneration and glaucoma are the leading causes of blindness worldwide. Automatic methods for diagnosis exist, but their performance is limited by the quality of the data. Spectral retinal images provide a significantly better representation of the colour information than common grayscale or red-green-blue retinal imaging, having the potential to improve the performance of automatic diagnosis methods. This work studies the image processing techniques required for composing spectral retinal images with accurate reflection spectra, including wavelength channel image registration, spectral and spatial calibration, illumination correction, and the estimation of depth information from image disparities. The composition of a spectral retinal image database of patients with diabetic retinopathy is described. The database includes gold standards for a number of pathologies and retinal structures, marked by two expert ophthalmologists. The diagnostic applications of the reflectance spectra are studied using supervised classifiers for lesion detection. In addition, inversion of a model of light transport is used to estimate histological parameters from the reflectance spectra. Experimental results suggest that the methods for composing, calibrating and postprocessing spectral images presented in this work can be used to improve the quality of the spectral data. The experiments on the direct and indirect use of the data show the diagnostic potential of spectral retinal data over standard retinal images. The use of spectral data could improve automatic and semi-automated diagnostics for the screening of retinal diseases, for the quantitative detection of retinal changes for follow-up, clinically relevant end-points for clinical studies and development of new therapeutic modalities.

Transhumanisme et Cellules Souches : travail à la frontière de la gériatrie biomédicale

La recherche scientifique biomédicale dans le domaine des cellules souches et plus largement de la médecine régénérative offre aujourd’hui des promesses d’applications thérapeutiques révolutionnaires pour de nombreuses maladies. Pourtant, il semble que pour certains, ces avancées pourraient servir d’autres desseins, notamment en ce qui concerne l’amélioration biologique de l’humain vers des objectifs de contrôle voire d’inversion du processus de vieillissement. Beaucoup de ceux qui tiennent à ces idées appartiennent à un mouvement, dit transhumaniste, où ils s’accordent sur des idées et valeurs communes concernant l’avenir de l’humain. Plus que cela, certains de ces acteurs transhumanistes prennent activement part à la recherche scientifique et orientent celle-ci vers les valeurs qu’ils soutiennent, touchant ainsi aux frontières de disciplines scientifiques établies et à la démarcation entre science et pseudoscience. En s’appuyant sur les concepts de recherche confinée / recherche de plein air, de forum hybride et de travail aux frontières, la présente recherche explore la place que les chercheurs transhumanistes occupent dans la recherche scientifique institutionnelle et se questionne sur la façon et les moyens qu’ils mettent en oeuvre pour y prendre part. À partir de la constitution et de l’analyse d’un corpus documentaire transhumaniste sur les cellules souches, mais aussi en décrivant le réseau auquel les chercheurs transhumanistes appartiennent, l’étude apporte une perspective nouvelle sur le mouvement transhumaniste. Les résultats obtenus montrent que les chercheurs transhumanistes ne se cantonnent pas à produire des discours et des représentations de leurs idées et de leurs valeurs, mais participent activement à la réalisation de celles-ci en menant eux-mêmes des recherches et en infiltrant la recherche scientifique institutionnelle.

Études fonctionnelles de deux nouvelles protéines centrosomales, NPHP5 et Cep76, et leurs implications dans les maladies humaines

Les centrosomes sont de petits organites qui régulent divers processus cellulaires comme la polarité ou la mitose dans les cellules de mammifères. Ils sont composés de deux centrioles entourés par une matrice péricentriolaire. Ces centrosomes sont les principaux centres organisateurs de microtubules. De plus, ils favorisent la formation de cils, des protubérances sur la surface des cellules quiescentes qui sont critiques pour la transduction du signal. Une grande variété de maladies humaines telles que les cancers ou les ciliopathies sont liées à un mauvais fonctionnement des centrosomes et des cils. C’est pourquoi le but de mes projets de recherche est de comprendre les mécanismes nécessaires à la biogénèse et au fonctionnement des centrosomes et des cils. Tout d'abord, j’ai caractérisé une nouvelle protéine centrosomale nommée nephrocystine - 5 (NPHP5). Cette protéine est localisée dans les cellules en interphase au niveau de la région distale des centrioles. Sa déplétion inhibe la migration des centrosomes à la surface cellulaire lors de l’étape précoce de la formation des cils. NPHP5 interagit avec la protéine CEP290 via sa région C-terminale qui est essentielle pour la ciliogenèse. Elle interagit également avec la calmoduline ce qui empêche son auto-agrégation. J’ai démontré que les domaines de liaison de NHPH5 à CEP290 et à la calmoduline, ainsi que son domaine de localisation centrosomale sont séparables. De plus, j’ai démontré que les protéines NPHP5 présentant des mutations pathogènes ne peuvent plus interagir avec CEP290 et ne sont plus localisées aux centrosomes, rendant ainsi ces protéines non fonctionnelles. Enfin, en utilisant une approche pharmacologique pour moduler les événements en aval dans la voie ciliogénique, j’ai montré que la formation des cils peut être restaurée même en absence de NPHP5. D’autre part, j’ai étudié le rôle de NPHP5 dans l'assemblage et le trafic du complexe BBSome dans le cil. Le BBSome est composé de huit sous-unités différentes qui s’assemblent en un complexe fonctionnel dont on sait peu de chose sur la régulation spatiotemporelle de son processus d'assemblage. J’ai précédemment montré que NPHP5 favorisait la formation des cils et que son dysfonctionnement contribuait au développement de néphronophtise (NPHP). Bien que la NPHP et le syndrome de Bardet-Biedl (BBS) soient des ciliopathies qui partagent des caractéristiques cliniques communes, la base moléculaire de ces ressemblances phénotypiques n’est pas comprise. J’ai constaté que NPHP5, localisé à la base du cil, contient deux sites de liaison distincts pour le BBSome. De plus, j’ai démontré que NPHP5 et son partenaire CEP290 interagissent de façon dynamique avec le BBSome pendant la transition de la prolifération à la quiescence. La déplétion de NPHP5 ou CEP290 conduit à la dissociation d’au moins deux sous-unités du BBSome formant alors un sous-complexe dont la capacité de migration dans le cil n’est pas compromise. J’ai montré que le transport des cargos vers le compartiment ciliaire par ce sous-complexe n’est que partiellement altéré. Enfin, j’ai également concentré mes recherches sur une autre protéine centrosomale peu caractérisée. La protéine centrosomale de 76 kDa (Cep76) a été précédemment impliquée dans le maintien d’une duplication unique des centrioles par cycle cellulaire, et dans une interaction avec la kinase cycline-dépendante 2 (CDK2). Cep76 est préférentiellement phosphorylée par le complexe cycline A/CDK2 sur le site unique S83. Cet événement est essentiel pour supprimer l'amplification des centrioles en phase S. J’ai démontré que Cep76 inhibe cette amplification en bloquant la phosphorylation de Plk1 au niveau des centrosomes. D’autre part, Cep76 peut être acétylée au site K279 en phase G2, ce qui régule négativement son activité et sa phosphorylation sur le site S83. Ces études permettent d'améliorer notre compréhension de la biologie des centrosomes et des cils et pourraient conduire au développement de nouvelles applications diagnostiques et thérapeutiques.

Purification and Characterization of an Anti-cancer Enzyme Produced by Marine Vibrio Costicola Under A Novel Solid State Fermentation Process

L - Glutaminase, a therapeutically and industrially important enzyme, was produced from marine Vibrio costicola by a novel solid state fermentation process using polystyrene beads as inert support. The new fermentation system offered several advantages over the conventional systems, such as the yield of leachate with minimum viscosity and high specific activity for the target product besides facilitating the easy estimation of biomass. The enzyme thus produced was purified and characterised. It was active at physiological pH, showed high substrate specificity towards L - glutamine and had a Km value of 7.4 x 10-2 M. It also exhibited high salt and temperature tolerance indicating good scope for its industrial and therapeutic applications

Soft Computing Approach for Optimization of siRNA Efficiency Prediction for Post-transcriptional Gene Silencing

Post-transcriptional gene silencing by RNA interference is mediated by small interfering RNA called siRNA. This gene silencing mechanism can be exploited therapeutically to a wide variety of disease-associated targets, especially in AIDS, neurodegenerative diseases, cholesterol and cancer on mice with the hope of extending these approaches to treat humans. Over the recent past, a significant amount of work has been undertaken to understand the gene silencing mediated by exogenous siRNA. The design of efficient exogenous siRNA sequences is challenging because of many issues related to siRNA. While designing efficient siRNA, target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. So before doing gene silencing by siRNAs, it is essential to analyze their off-target effects in addition to their inhibition efficiency against a particular target. Hence designing exogenous siRNA with good knock-down efficiency and target specificity is an area of concern to be addressed. Some methods have been developed already by considering both inhibition efficiency and off-target possibility of siRNA against agene. Out of these methods, only a few have achieved good inhibition efficiency, specificity and sensitivity. The main focus of this thesis is to develop computational methods to optimize the efficiency of siRNA in terms of “inhibition capacity and off-target possibility” against target mRNAs with improved efficacy, which may be useful in the area of gene silencing and drug design for tumor development. This study aims to investigate the currently available siRNA prediction approaches and to devise a better computational approach to tackle the problem of siRNA efficacy by inhibition capacity and off-target possibility. The strength and limitations of the available approaches are investigated and taken into consideration for making improved solution. Thus the approaches proposed in this study extend some of the good scoring previous state of the art techniques by incorporating machine learning and statistical approaches and thermodynamic features like whole stacking energy to improve the prediction accuracy, inhibition efficiency, sensitivity and specificity. Here, we propose one Support Vector Machine (SVM) model, and two Artificial Neural Network (ANN) models for siRNA efficiency prediction. In SVM model, the classification property is used to classify whether the siRNA is efficient or inefficient in silencing a target gene. The first ANNmodel, named siRNA Designer, is used for optimizing the inhibition efficiency of siRNA against target genes. The second ANN model, named Optimized siRNA Designer, OpsiD, produces efficient siRNAs with high inhibition efficiency to degrade target genes with improved sensitivity-specificity, and identifies the off-target knockdown possibility of siRNA against non-target genes. The models are trained and tested against a large data set of siRNA sequences. The validations are conducted using Pearson Correlation Coefficient, Mathews Correlation Coefficient, Receiver Operating Characteristic analysis, Accuracy of prediction, Sensitivity and Specificity. It is found that the approach, OpsiD, is capable of predicting the inhibition capacity of siRNA against a target mRNA with improved results over the state of the art techniques. Also we are able to understand the influence of whole stacking energy on efficiency of siRNA. The model is further improved by including the ability to identify the “off-target possibility” of predicted siRNA on non-target genes. Thus the proposed model, OpsiD, can predict optimized siRNA by considering both “inhibition efficiency on target genes and off-target possibility on non-target genes”, with improved inhibition efficiency, specificity and sensitivity. Since we have taken efforts to optimize the siRNA efficacy in terms of “inhibition efficiency and offtarget possibility”, we hope that the risk of “off-target effect” while doing gene silencing in various bioinformatics fields can be overcome to a great extent. These findings may provide new insights into cancer diagnosis, prognosis and therapy by gene silencing. The approach may be found useful for designing exogenous siRNA for therapeutic applications and gene silencing techniques in different areas of bioinformatics.

The use of proteomics to identify novel therapeutic targets for the treatment of disease

The completion of the Human Genome Project has revealed a multitude of potential avenues for the identification of therapeutic targets. Extensive sequence information enables the identification of novel genes but does not facilitate a thorough understanding of how changes in gene expression control the molecular mechanisms underlying the development and regulation of a cell or the progression of disease. Proteomics encompasses the study of proteins expressed by a population of cells, and evaluates changes in protein expression, post-translational modifications, protein interactions, protein structure and splice variants, all of which are imperative for a complete understanding of protein function within the cell. From the outset, proteomics has been used to compare the protein profiles of cells in healthy and diseased states and as such can be used to identify proteins associated with disease development and progression. These candidate proteins might provide novel targets for new therapeutic agents or aid the development of assays for disease biomarkers. This review provides an overview of the current proteomic techniques available and focuses on their application in the search for novel therapeutic targets for the treatment of disease.

Therapeutic modulation of microbiota-host metabolic interactions

The complex metabolic relationships between the host and its microbiota change throughout life and vary extensively between individuals, affecting disease risk factors and therapeutic responses through drug metabolism. Elucidating the biochemical mechanisms underlying this human supraorganism symbiosis is yielding new therapeutic insights to improve human health, treat disease, and potentially modify human disease risk factors. Therapeutic options include targeting drugs to microbial genes or co-regulated host pathways and modifying the gut microbiota through diet, probiotic and prebiotic interventions, bariatric surgery, fecal transplants, or ecological engineering. The age-associated co-development of the host and its microbiota provides a series of windows for therapeutic intervention from early life through old age

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Natural gum-assisted phthalocyanine immobilization in electroactive nanocomposites: Physicochemical characterization and sensing applications

Natural gums have been traditionally applied in cosmetics and the food industry, mainly as emulsification agents. Due to their biodegradability and excellent mechanical properties, new technological applications have been proposed involving their use with conventional polymers forming blends and composites. In this study, we take advantage of the polyelectrolyte character exhibited by the natural gum Chicha (Sterculia striata), extracted in the Northeastern region of Brazil, to produce electroactive nanocomposites. The nanocomposites were fabricated in the form of ultrathin films by combining a metallic phthalocyanine (nickel tetrasulfonated phthalocyanine, NiTsPc) and the Chicha gum in a tetralayer architecture, in conjunction with conventional polyelectrolytes. The presence of the gum led to an efficient adsorption of the phthalocyanine and enhanced the electrochemical response of the films. Upon combining the electrochemical and UV-vis absorption data, energy diagrams of the Chicha/NiTsPc-based system were obtained. Furthermore, modified electrodes based on gum/phthalocyanine films were able to detect dopamine at concentrations as low as 10(-5) M.

Experimental Basis and New Insights for Cell Therapy in Chronic Obstructive Pulmonary Disease

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of Neural Stem/Progenitor Cells Expressing VEGF and its Receptors in the Subventricular Zone of Newborn Piglet Brain

Neural stem/progenitor cell (NSP) biology and neurogenesis in adult central nervous system (CNS) are important both towards potential future therapeutic applications for CNS repair, and for the fundamental function of the CNS. In the present study, we report the characterization of NSP population from subventricular zone (SVZ) of neonatal piglet brain using in vivo and in vitro systems. We show that the nestin and vimentin-positive neural progenitor cells are present in the SVZ of the lateral ventricles of neonatal piglet brain. In vitro, piglet NSPs proliferated as neurospheres, expressed the typical protein of neural progenitors, nestin and a range of well-established neurodevelopmental markers. Upon dissociation and subculture, piglet NSPs differentiated into neurons and glial cells. Clonal analysis demonstrates that piglet NSPs are multi-potent and retain the capacity to generate both glia and neurons. These cells expressed VEGF, VEGFR1, VEGFR2 and Neuropilin-1 and -2 mRNAs. Real time PCR revealed that SVZ NSPs from newborn piglet expressed total VEGF and all VEGF splice variants. These findings show that piglet NSPs may be helpful to more effectively design growth factor based strategies to enhance endogenous precursor cells for cell transplantation studies potentially leading to the application of this strategy in the nervous system disease and injury.

Design of novel iron compounds as potential therapeutic agents against tuberculosis

In the search for new therapeutic tools against tuberculosis two novel iron complexes, [Fe(L-H)3], with 3-aminoquinoxaline-2-carbonitrile N(1),N(4)-dioxide derivatives (L) as ligands, were synthesized, characterized by a combination of techniques, and in vitro evaluated. Results were compared with those previously reported for two analogous iron complexes of other ligands of the same family of quinoxaline derivatives. In addition, the complexes were studied by cyclic voltammetry and EPR spectroscopy. Cyclic voltammograms of the iron compounds showed several cathodic processes which were attributed to the reduction of the metal center (Fe(III)/Fe(II)) and the coordinated ligand. EPR signals were characteristic of magnetically isolated high-spin Fe(III) in a rhombic environment and arise from transitions between m(s) = +/- 1/2 (geff-9) or m(s) = +/- 3/2 (g(eff)similar to 4.3) states. Mossbauer experiments showed hyperfine parameters that are typical of high-spin Fe(III) ions in a not too distorted environment. The novel complexes showed in vitro growth inhibitory activity on Mycobacterium tuberculosis H(37)Rv (ATCC 27294), together with very low unspecific cytotoxicity on eukaryotic cells (cultured murine cell line J774). Both complexes showed higher inhibitory effects on M. tuberculosis than the "second-line" therapeutic drugs. (C) 2010 Elsevier B.V. All rights reserved.