Experimental Assessment of the Sensitiveness of an Electrochemical Oscillator towards Chemical Perturbations

In this study we address the problem of the response of a (electro)chemical oscillator towards chemical perturbations of different magnitudes. The chemical perturbation was achieved by addition of distinct amounts of trifluoromethanesulfonate (TFMSA), a rather stable and non-specifically adsorbing anion, and the system under investigation was the methanol electro-oxidation reaction under both stationary and oscillatory regimes. Increasing the anion concentration resulted in a decrease in the reaction rates of methanol oxidation and a general decrease in the parameter window where oscillations occurred. Furthermore, the addition of TFMSA was found to decrease the induction period and the total duration of oscillations. The mechanism underlying these observations was derived mathematically and revealed that inhibition in the methanol oxidation through blockage of active sites was found to further accelerate the intrinsic non-stationarity of the unperturbed system. Altogether, the presented results are among the few concerning the experimental assessment of the sensitiveness of an oscillator towards chemical perturbations. The universal nature of the complex chemical oscillator investigated here might be used for reference when studying the dynamics of other less accessible perturbed networks of (bio)chemical reactions.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

The phospholipases A(1) (PLA(1)s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1), combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2)s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa.

PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.

Blood-Borne Circadian Signal Stimulates Daily Oscillations in Actin Dynamics and SRF Activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

Étude des mécanismes d’inactivation des microorganismes suite à un traitement à l’ozone

Une partie du travail a mené a un dépôt de brevet.

Développement d'une méthode de quantification des varicosités synaptiques et asynaptiques établies par les neurones dopaminergiques

Les neurones dopaminergiques (DA) de la substance noire compacte (SNc) et de l’aire tegmentaire ventrale (ATV) développent des contacts de type synaptique et non synaptique. Malgré de nombreux travaux sur la synaptogénèse en général, aucune méthode autre que la microscopie électronique, n’a été développée pour quantifier les varicosités synaptiques et asynaptiques issues des neurones DA. L’objectif principal de ce projet était de développer une méthode d’analyse et de quantification des varicosités synaptiques et asynaptiques des neurones DA. L’hypothèse proposée est qu’il devait être possible de détecter la présence de synapses en visualisant la colocalisation d’une protéine présynaptique telle que synaptotagmine 1 (SYT1) avec un marqueur post-synaptique tel que la postsynaptic density protein 95 (PSD95). Pour ce faire, nous avons préparé des cultures primaires de neurones DA à l’aide d’une lignée de souris transgéniques exprimant la protéine fluorescente verte (GFP) sous le contrôle du promoteur de la tyrosine hydroxyalse (TH). Nous avons ensuite visualisé les terminaisons axonales à l'aide de marquages immunocytochimiques de protéines pré et post-synaptiques. L’analyse quantitative des images a été effectuée avec le logiciel de traitement d’image Image-J. Nos résultats montrent que, via l’association d’un marqueur présynaptique tel que SYT1 avec un marqueur postsynaptique tel que PSD95, seule une minorité des terminaisons établies par les neurones DA sont de type synaptique. En contraste, des neurones glutamatergiques du cortex, établissent une majorité de terminaisons associées à un marqueur postsynaptique. Nos résultats valident donc la mise en place d'une technique d'analyse permettant de quantifier la proportion de terminaisons synaptiques et asynaptiques établies par les neurones DA.

Effect of oxidized low-density lipoprotein on differential gene expression in primary human endothelial cells

Oxidative modification of low-density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis. It has been proposed that the biological action of oxidized LDL (ox-LDL) may be partially attributed to its effect on a shift of the pattern of gene expression in endothelial cells. To examine the transcriptional response to ox-LDL, we applied cDNA array technology to cultured primary human endothelial cells challenged with oxidized human LDL. A twofold or greater difference in the expression of a particular gene was considered a significant difference in transcript abundance. Seventy-eight of the 588 genes analyzed were differentially expressed in response to the treatment. Ox-LDL significantly affected the expression of genes encoding for transcription factors, cell receptors, growth factors, adhesion molecules, extracellular matrix proteins, and enzymes involved in cholesterol metabolism. The alteration of the expression pattern of several genes was substantiated post hoc using RT-PCR. The experimental strategy identified several novel ox-LDL-sensitive genes associated with a "response to injury" providing a conceptual background to be utilized for future studies addressing the molecular basis of the early stages of atherogenesis.

Identification of hepatic molecular mechanisms of action of alpha-tocopherol using global gene expression profile analysis in rats

The recent discovery that vitamin E (VE) regulates gene activity at the transcriptional level indicates that VE may exert part of its biological effects by mechanisms which may be independent of its well-recognised antioxidant function. The objective of this study was the identification of hepatic vitamin E-sensitive genes and examination of the effects of VE on their corresponding biological endpoints. Two groups of male rats were randomly assigned to either a VE-sufficient diet or to a control diet deficient in VE for 290 days. High-density oligonucleotide microarrays comprising over 7000 genes were used to assess the transcriptional response of the liver. Differential gene expression was monitored over a period of 9 months, at four different time-points, and rats were individually profiled. This experimental strategy identified several VE-sensitive genes, which were chronically altered by dietary VE. VE supplementation down-regulated scavenger receptor CD36, coagulation factor IX and 5-alpha-steroid reductase type 1 mRNA levels while hepatic gamma glutamyl-cysteinyl synthetase was significantly up-regulated. Measurement of the corresponding biological endpoints such as activated partial thromboplastin time, plasma dihydrotestosterone and hepatic glutathione substantiated the gene chip data which indicated that dietary VE plays an important role in a range of metabolic processes within the liver. (C) 2004 Elsevier B.V. All rights reserved.

Proteomic characterization of the multiple forms of the PLAs from the venom of the social wasp Polybia paulista

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

Uma contribuição para a otimização de portfólios de séries heteroscedásticas usando projeto de experimento de misturas: uma abordagem do desirability aplicada a modelos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro

Abstract Background Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans. Results Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings. Conclusion Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.

STUDYING AGGREGATE FORMATION BY AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTANT SOD1 PROTEIN IN DROSOPHILA MODEL

A common pathological hallmark of most neurodegenerative disorders is the presence of protein aggregates in the brain. Understanding the regulation of aggregate formation is thus important for elucidating disease pathogenic mechanisms and finding effective preventive avenues and cures. Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a selective neurodegenerative disorder predominantly affecting motor neurons. The majority of ALS cases are sporadic, however, mutations in superoxide dismutase 1 (SOD1) are responsible for about 20% of familial ALS (fALS). Mutated SOD1 proteins are prone to misfold and form protein aggregates, thus representing a good candidate for studying aggregate formation. The long-term goal of this project is to identify regulators of aggregate formation by mutant SOD1 and other ALS-associated disease proteins. The specific aim of this thesis project is to assess the possibility of using the well-established Drosophila model system to study aggregation by human SOD1 (hSOD1) mutants. To this end, using wild type and the three mutant hSOD1 (A4V, G85R and G93A) most commonly found among fALS, I have generated 16 different SOD1 constructs containing either eGFP or mCherry in-frame fluorescent reporters, established and tested both cell- and animal-based Drosophila hSOD1 models. The experimental strategy allows for clear visualization of ectopic hSOD1 expression as well as versatile co-expression schemes to fully investigate protein aggregation specifically by mutant hSOD1. I have performed pilot cell-transfection experiments and verified induced expression of hSOD1 proteins. Using several tissue- or cell type-specific Gal4 lines, I have confirmed the proper expression of hSOD1 from established transgenic fly lines. Interestingly, in both Drosophila S2 cells and different fly tissues including the eye and motor neurons, robust aggregate formation by either wild type or mutant hSOD1 proteins was not observed. These preliminary observations suggest that Drosophila might not be a good experimental organism to study aggregation and toxicity of mutant hSOD1 protein. Nevertheless this preliminary conclusion implies the potential existence of a potent protective mechanism against mutant hSOD1 aggregation and toxicity in Drosophila. Thus, results from my SOD1-ALS project in Drosophila will help future studies on how to best employ this classic model organism to study ALS and other human brain degenerative diseases.

Visualization of biochemical networks in living cells

Functional annotation of novel genes can be achieved by detection of interactions of their encoded proteins with known proteins followed by assays to validate that the gene participates in a specific cellular function. We report an experimental strategy that allows for detection of protein interactions and functional assays with a single reporter system. Interactions among biochemical network component proteins are detected and probed with stimulators and inhibitors of the network. In addition, the cellular location of the interacting proteins is determined. We used this strategy to map a signal transduction network that controls initiation of translation in eukaryotes. We analyzed 35 different pairs of full-length proteins and identified 14 interactions, of which five have not been observed previously, suggesting that the organization of the pathway is more ramified and integrated than previously shown. Our results demonstrate the feasibility of using this strategy in efforts of genomewide functional annotation.

Identification of the coding region for a second poly(A) polymerase in Escherichia coli.

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.

Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA.DNA oligonucleotide.

An experimental strategy to facilitate correction of single-base mutations of episomal targets in mammalian cells has been developed. The method utilizes a chimeric oligonucleotide composed of a contiguous stretch of RNA and DNA residues in a duplex conformation with double hairpin caps on the ends. The RNA/DNA sequence is designed to align with the sequence of the mutant locus and to contain the desired nucleotide change. Activity of the chimeric molecule in targeted correction was tested in a model system in which the aim was to correct a point mutation in the gene encoding the human liver/bone/kidney alkaline phosphatase. When the chimeric molecule was introduced into cells containing the mutant gene on an extrachromosomal plasmid, correction of the point mutation was accomplished with a frequency approaching 30%. These results extend the usefulness of the oligonucleotide-based gene targeting approaches by increasing specific targeting frequency. This strategy should enable the design of antiviral agents.