The current knowledge concerning effects of polybrominated diphenyl ether (PBDEs) on benthic aquatic organisms is still very limited although they have been widely used as fire retardant additives for 3 decades. This study was conducted to evaluate the toxic effects of BDE-47 and BDE-183, the two common congeners of PBDEs in river sediments, on a benthic oligochaete worms, Monopylephorus limosus. The worms were exposed to BDE-47 or to BDE-183 for two or eight weeks. The survival rates of M. limosus decreased significantly when exposed to 700 ng/g dry soil of BDE-47 or BDE-183 for 8 weeks, but not in groups of 1-1000 ng/g BDE-47 for 2 weeks. A total of forty proteins of M. limosus has been expressed and determined by the two-dimensional gel electrophoresis (2-DE). Through the cluster analysis, it was found that the protein expression in the group of 100 ng/g BDE-47 was similar to 10 ng/g BDE-183. The results indicate that the toxicity of BDE-183 was greater than BDE-47 to M.limosus. Although the survival rate of M. limosus was not significantly affected when exposed to BDE-47 or BDE-183 at concentrations of 1 to 100 ng/g, significant differences in protein expression were found. Thus, the analysis of protein expression is more sensitive to detect the toxicological change in M. limosus than the survival test.

Gene of DC2 protein, a novel unknown gene, was identified previously in our laboratory while studying the death progression in the rat brain stem. According to the search results of bioinformatics database, both human DC2 and house mouse DC2 are 149 amino acids long and 16.8 kDa. The entire sequence of human DC2 differs from house mouse DC2 by only a single amino acid substitution. The bioinformatics revealed that human DC2 and house mouse DC2 had three predicted transmembrane regions. These results suggest human DC2 and house mouse are highly homologous. DC2 protein expresses differentially between organs. Human liver is the top fourth DC2-expressed organ, while house mouse liver is ranked 23rd DC2-expressed organ. Shibatani et al (Shibatani et al¡A2005) proposed DC2 protein as a potential subunit of mammalian Oligosaccharyltransferase (OST) after mass spectrometry analysis and suggested DC2 might involve in glycosylation. House mouse liver fibrosis was induced by giving 300mg/L thioacetamide (TAA) in the drinking water for different periods of time, and then gene expression of house mouse DC2 of liver was analyzed. mRNA expression was found in normal house mouse liver and mRNA expression increased gradually after TAA administration. DC2 protein also found in normal house mouse liver and DC2 protein of house mouse liver increased after TAA administration.

In tilapia , sex determination is controled by genetic and triggered by the environmental factors. Expressed sequence tags ( EST ) derived from the developing tilapia brain is cloned in our lab. The cDNA full length of the gene, F10A83 was cloned. In the present study, F10A83 is a gene with 1526 bp of cDNA sequence, and deduced 176 amino acids of protein sequence. F10A83 was overexpressed as a GFP fusion protein in mouse neuroblastoma Neuro-2a cells. F10A83 is abundantly distributed in the nucleus of Neuro-2a cell. The protein of F10A83 was expressed in the prokaryotic cell (BL21) and eukaryotic cell (neuro-2a), and purified using a simple purification process, inducing, isolation, and Ni-NTA affinity chromatography. The protein of F10A83 in both E. coli system and neuro-2a cell line expression has been established.

Steroid hormones, such as estrogen, are produced in one tissue and carried through the blood stream to target tissues in which they bind to highly specific nuclear receptors and trigger changes in gene expression and metabolism. Industrial chemicals, such as bisphenol A and many agricultural chemicals, including permethrin and fervalerate, are known to have estrogenic potential and therefore are estrogen mimics. Widely used agricultural chemicals, Enable (fungicide) and Diazinon (insecticide), were evaluated to examine their toxicity and estrogenicity. MCF-7 cells, an estrogen-dependent human breast cancer line, were utilized for this purpose. MCF-7 cells were treated with 0.033-3.3 ppb (ng/ml) of Enable and 0.3-67 ppm of Diazinon and gene expression was compared to that in untreated cells. Microarray analysis showed down-regulation of eight genes and up-regulation of thirty four genes in cells treated with 3.3 ppb of Enable, compared to untreated cells. Similarly, in cells treated with 67 ppm of Diazinon, there were three genes down-regulated and twenty seven genes up-regulated. For both chemicals, specific genes were selected for special consideration. RT-PCR confirmed results obtained from analysis of the microarray. These studies were designed to provide base-line data on gene expression-altering capacity of specific chemicals and will allow assessment of the deleterious effects caused by exposure to the aforementioned chemicals.

The horned condition in cattle is believed to be the wild type with morphogenesis primarily occurring after birth. The polled condition has existed since domestication and has been selected for its economic importance. The polled locus has previously been mapped by genetic linkage analysis to the proximal region of bovine chromosome 1. In order to help us eventually identify the causative mutation, the objective of the study was to structurally and functionally characterize the polled interval from IFNAR1 to SOD1 on BTA1. Our hypothesis was that the polled locus is a tissue specific transcription factor that is expressed in the developing horn buds and acts directly or indirectly upon SOX9. A 2.5 Mb BAC contig and STS content map of the polled interval was constructed. Three candidate genes encoding transcription factors were identified within this region but only C21orf66 was expressed in the horn buds from 1 d old Bos indicus influenced calves. The C21orf66 gene has 18 exons, spans 30,976 bp of genomic DNA, and 144 SNP were identified. No single SNP discovered in C21orf66 can be attributed as the causative mutation. None of the genes from the polled interval were differentially expressed in skin and horn from 1 d old Bos indicus influenced calves. However, there were significant differences in the levels of expression of RUNX2, SOX9, BMP4, PRKCA, and FOXL2 in these samples. Expression of RUNX2 was localized to the osteoblasts, and both RUNX2 and SOX9 were expressed in sebaceous glands of the horn at 1 d of age. Histological examination of horns and scurs from newborn, 5 to 6 mo, and ~1.5 yr old Bos indicus influenced cattle suggest that horns form through intramembranous ossification. Based on the data presented herein, we propose that the polled locus is upstream of RUNX2 and SOX9 in the osteogenic pathway, and could have its primary effect on the differentiation of mesenchymal condensations. The genes IL10RB, SFRS15, C21orf66, OLIG1, OLIG2 and HUNK remain candidates for the polled locus and warrant further investigation.

<p>Today one of the greatest aims within the area of bioinformatics is to gain a complete understanding of the functionality of genes and the systems behind gene regulation. Regulatory relationships among genes seem to be of a complex nature since transcriptional control is the result of complex networks interpreting a variety of inputs. It is therefore essential to develop analytical tools detecting complex genetic relationships.</p><p>This project examines the possibility of the data mining technique artificial neural network (ANN) detecting regulatory relationships between genes. As an initial step for finding regulatory relationships with the help of ANN the goal of this project is to train an ANN to predict the expression of an individual gene. The genes predicted are the nuclear receptor PPAR-g and the insulin receptor. Predictions of the two target genes respectively were made using different datasets of gene expression data as input for the ANN. The results of the predictions of PPAR-g indicate that it is not possible to predict the expression of PPAR-g under the circumstances for this experiment. The results of the predictions of the insulin receptor indicate that it is not possible to discard using ANN for predicting the gene expression of an individual gene.</p>

<p>The innovation of DNA analysis techniques has enabled arevolution in the field of molecular biology. In the 70’s,first technologies for sequence determination of DNA wereinvented and these techniques enormously increased thepossibilities of genetic research. A large proportion ofmethods for DNA sequencing is based on enzymatic DNA synthesiswith chain termination followed by electrophoretic separationand detection. However, alternative approaches have beendeveloped and one example of this is the pyrosequencingtechnology, which a four-enzyme DNA sequencing method based onreal-time monitoring of DNA synthesis.</p><p>Currently, the method is limited to analysis of short DNAsequences and therefore it has primarily been used for mutationdetection and single-nucleotide polymorphism analysis. In orderto expand the use of the pyrosequencing technology, the readlength obtained in the methods needs to be improved. However,it was previously shown that the data quality in pyrosequencingtechnology could be significantly increased by addition ofEscherichia coli single-stranded DNAbinding protein, SSB, tothe sequencing reaction. Since little was known about themechanism of this enhancement, we performed a systematic effortto analyse the effect of SSB on 103 clones randomly selectedfrom a cDNA library. We investigated the effect of SSB on theobtained read length in pyrosequencing and identified thecauses of low quality sequences. Moreover, the effciency ofprimer annealing and SSB binding for individual cDNA clones wasinvestigated by use of real-time biosensor analysis. Resultsfrom these experiments show that templates with highperformance in pyrosequencing without SSB possess effcientprimer annealing and low SSB affnity.</p><p>To minimise the cost of the pyrosequencing system, effcientand scaleable procedures for production and isolation of theprotein components are required. Therefore, protocol foreffcient expression in E.<i>coli</i>and rapid isolation of native SSB was developed.Moreover, by use of a gene fusion strategy, Klenow polymerasewas produced in fusion with the Zbasic domain at high levels inE. coli. This highly charged protein handle enables selectiveand effcient ion exchange purification at physiological pH.Furthermore, active Apyrase was expressed in Methyltropic yeastPichia pastoris and purified by two chromatographic steps.</p><p>Since pyrosequencing analysis mainly is performed in a96-sample plate format, an increase in sample capacity would bevery beneficial. One approach to achieve this would be to usemicromachined filter chamber arrays where nano-liter samplescan be monitored in real-time. However, to enable accuratepyrosequencing analysis of parallel samples, the produced lightshould preferable be docked to the correct DNA template.Therefore, two different gene fusion strategies were utilisedbased on directed immobilisation of the light-harvesting enzymeLuciferase on the DNA molecules. The thermostable variant ofthe enzyme was genetically fused to a DNA binding protein(either SSB or Klenow) and the Z_basic purification handle, which could beselectively removed by protease cleavage. A protocol wasdeveloped for effcient expression in E.<i>coli</i>and purification by Ion Exchange Chromatography.The proteins were analysed by complete extension of DNAtemplates immobilised on magnetic beadspyrosequencing monitoredby pyrosequencing chemistry. Results from these experimentsshow that the proteins bound selectively to the immobilised DNAand that their enzymatic domains were active.</p><p>In summary, the work presented in this thesis pinpointsfeatures in the pyrosequencing technology that needs to befurther developed. Moreover, various protein-based strategiesare presented in order to overcome these limitations.</p><p><b>Keywords:</b>pyrosequencing, SSB, Z_basic, Klenow, Apyrase, expression, purification,Biacore, DNA template length, Luciferase, affnity, gene fusion,immobilisation.</p>

<p>Gene expression analysis has evolved as an extensive toolfor elucidation of various biological and molecular eventsoccurring in different organisms. A variety of techniques andsoftware tools have been developed to enable easier and morerapid means of exploring the genetic information. A moreeffective approach than exploring the whole content of genesexpressed under certain conditions is to study fingerprintassays or to use subtracted cDNA libraries to identify onlydifferentially expressed genes.</p><p>The objective for the work in this thesis has been toexplore differentially expressed genes in atherosclerosis. Thiswas done by applying and modifying a protocol for thesubtractive approach RDA (Representational Difference Analysis)in different model systems.</p><p>Initially, the molecular effects of an anti-atheroscleroticdrug candidate were elucidated. In addition, two alternativeapproaches to identify differentially expressed genes obtainedafter iterative rounds of RDA subtraction cycles wereevaluated. This revealed that in most cases, the shotgunapproach in which the obtained gene fragments are clonedwithout any prior selection has clear advantages compared tothe more commonly used selection strategy, whereby distinctbands are excised after gel electrophoresis.</p><p>A key process in the atherosclerotic plaque initiation isthe phenotypic change of macrophages into foam cells, which canbe triggered in a model system by using macrophages exposed tooxidised LDL. To investigate the genes expressed in thisprocess, the RDA technique was combined with microarrayanalysis, which allows for selectivity and sensitivity throughRDA, as well as rapid high-throughput analysis usingmicroarrays. The combination of these techniques enablessignificant differences in gene expression to be detected, evenfor weakly expressed genes and the results to be reliablyvalidated in a high throughput manner.</p><p>Finally, investigation of the focal nature ofatherosclerotic lesions and gene expression profiling werestudied using in vivo aortic tissues from ApoE-/- and LDLR -/-mice. The study was based on a comparison between localisationsthat are likely, and others that are unlikely, to developatherosclerotic plaques, and the RDA technique was employed toexplore differential gene expression.</p><p><b>Keywords:</b>Representational Difference Analysis,atherosclerosis, gene expression profiling</p>

An experienced dermatopathologist can reliably diagnose most cutaneous malignant melanomas based on well-established morphologic characteristics. However, in a minority of cases, traditional histopathologic evaluation and immunohistochemistry (IHC) are inadequate to confidently distinguish melanoma from benign melanocytic lesions such as Spitz nevi and dysplastic nevi. The advent of high-throughput gene expression array technology has resulted in the identification of hundreds of potential molecular diagnostic biomarkers, but no single chromosomal, DNA, RNA or protein marker has yet been shown to differentiate melanoma from nevus with sensitivity and specificity approaching 100%. We selected the protein products of 11 genesATP1B1, CYCLIN D1, DLX-1, HOXB13, LIF, MEIS2, MITF, MYC, PHACTR1, PTPRF and TWISTup-regulated in melanoma cell-culture and tissue-based expression arrays as candidate diagnostic biomarkers for preliminary investigation. Based on the results of our pilot studies, we proposed that increased expression of Phactr1 protein, as measured by IHC, could be used to differentiate malignant melanomas from nevi. We applied Phactr1 monoclonal antibody to a 480-core tissue microarray that included samples from 28 nevi and 62 primary melanomas. A simple scoring algorithm derived from this data distinguished primary melanoma from nevus in the training set with 92% sensitivity and 100% specificity. These data suggest that Phactr1 immunohistochemical staining is a potentially useful aid in the clinical diagnosis of primary cutaneous melanoma.

Texte remanié de: Th. doct.--Droit privé--Paris 1, 2002. / Bibliogr. p. 329-353. Index.